CN113501880A - Compositions and methods relating to engineered Fc constructs - Google Patents

Compositions and methods relating to engineered Fc constructs Download PDF

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CN113501880A
CN113501880A CN202110761899.9A CN202110761899A CN113501880A CN 113501880 A CN113501880 A CN 113501880A CN 202110761899 A CN202110761899 A CN 202110761899A CN 113501880 A CN113501880 A CN 113501880A
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domain
construct
domain monomer
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val
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C·J·博斯克斯
J·S·休斯顿
J·C·兰星
L·E·凌
J·米多尔
D·奥尔蒂斯
L·鲁蒂茨基
B·C·舒尔特斯
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Momenta Pharmaceuticals Inc
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Abstract

The present invention relates to compositions and methods for engineering IgG Fc constructs, wherein the Fc construct comprises one or more Fc domains.

Description

Compositions and methods relating to engineered Fc constructs
This application is a divisional application of chinese patent application 201580020385.0 "compositions and methods relating to engineered Fc constructs" filed on day 2015, 5, month 1.
Background
Therapeutic proteins, such as therapeutic antibodies and Fc-fusion proteins, have rapidly become a clinically important class of drugs for patients with immunological and inflammatory diseases.
Disclosure of Invention
The present invention relates to biologically active Fc domain-containing therapeutic constructs. Such constructs may have a desirable serum half-life and/or binding affinity and/or avidity for Fc receptors. These constructs may be useful, for example, to reduce inflammation in a subject, promote autoantibody clearance in a subject, inhibit antigen presentation in a subject, block an immune response in a subject (e.g., block the immune response based on activation of an immune complex), and treat immunological and inflammatory diseases in a subject (e.g., autoimmune diseases). The Fc constructs described herein can be used to treat patients with immunological and inflammatory diseases without the need for significant stimulation of immune cells.
In general, the invention relates to Fc constructs having 2-10 Fc domains, e.g., Fc constructs having 2, 3, 4, 5, 6, 7, 8, 9, or 10 Fc domains. In some embodiments, the Fc construct comprises 2-10 Fc domains, 2-5 Fc domains, 3-5 Fc domains, 2-8 Fc domains, or 2-6 Fc domains. The construct may comprise 2-6 (e.g., 2, 3, 4, 5, or 6) related polypeptides, each polypeptide comprising at least one Fc domain monomer, wherein each Fc domain monomer of the construct is the same as or differs by no more than 20 amino acids (e.g., no more than 15, 10 amino acids), such as no more than 20, 15, 10, 8, 7, 6, 5, 4, 3, or 2 amino acids, from another monomer of the construct. The Fc constructs described herein do not comprise an antigen binding domain of an immunoglobulin. In some embodiments, the Fc construct (or Fc domain within the Fc construct) is formed, in whole or in part, by association of Fc domain monomers present in different polypeptides. In certain embodiments, the Fc construct does not comprise an additional domain (e.g., an IgM tail or an IgA tail) that facilitates association of the two polypeptides. In other embodiments, the covalent bond is only present between two Fc domain monomers linked to form an Fc domain in the Fc construct. In other embodiments, the Fc construct does not comprise covalent bonds between Fc domains. In still other embodiments, the Fc construct provides sufficient structural flexibility such that all or substantially all of the Fc domains in the Fc construct are capable of interacting with Fc receptors on the surface of a cell simultaneously. In some embodiments, the Fc construct comprises at least two Fc domains connected by a linker (e.g., a flexible amino acid spacer). In one embodiment, the primary sequences of the domain monomers differ from wild-type or each other in that they have a dimerization selectivity module.
The Fc constructs of the invention can be in a pharmaceutical composition comprising a substantially homologous set (e.g., at least 85%, 90%, 95%, 98%, or 99% homologous) of Fc constructs having 2-10 Fc domains (e.g., constructs having 2, 3, 4, 5, 6, 7, 8, 9, or 10 Fc domains, such as those described herein). Thus, pharmaceutical compositions of Fc constructs can be produced that do not have substantial aggregation or unwanted multimerization.
In one aspect, the Fc construct comprises three polypeptides forming two Fc domains. The first polypeptide has the formula a-L-B, wherein a comprises a first Fc domain monomer; l is a linker; and B comprises a second Fc domain monomer. The second polypeptide comprises a third Fc domain monomer, and the third polypeptide comprises a fourth Fc domain monomer. In this aspect, the first Fc domain monomer and the third Fc domain monomer combine to form the first Fc domain. Similarly, the second Fc domain monomer and the fourth Fc domain monomer combine to form a second Fc domain. Exemplary Fc constructs of this aspect of the invention are shown in fig. 4 and 6.
In certain embodiments, the first Fc domain monomer and the third Fc domain monomer comprise complementary dimerization selectivity modules that promote dimerization between the Fc domain monomers. In other embodiments, the second Fc domain monomer and the fourth Fc domain monomer comprise complementary dimerization selectivity modules that promote dimerization between the Fc domain monomers.
In certain embodiments, one or more of A, B, the second polypeptide, and the third polypeptide consists of an Fc domain monomer. In one embodiment, A, B, the second polypeptide, and the third polypeptide each consist of an Fc domain monomer.
In certain embodiments, the Fc construct may further comprise a heterologous moiety, such as a peptide, e.g., a serum protein-binding peptide, e.g., an albumin-binding peptide. This moiety may be attached to the N-terminus or the carboxy-terminus of B or the third polypeptide, for example, by a linker.
In certain embodiments, the Fc construct further comprises an IgG CLAntibody constant domains and an IgG C H1 antibody constant domain. IgG C H1 the antibody constant domain may be attached to the N-terminus of the a or second polypeptide, e.g., by a linker.
In other embodiments, the second and third polypeptides of the Fc construct have the same amino acid sequence.
In another aspect, the invention relates to an Fc construct comprising four polypeptides forming three Fc domains. The first polypeptide has the formula a-L-B, wherein a comprises a first Fc domain monomer; l is a linker; and B comprises a second Fc domain monomer. The second polypeptide has the formula a '-L' -B ', wherein a' comprises a third Fc domain monomer; l' is a linker; and B' comprises a fourth Fc domain monomer. The third polypeptide comprises a fifth Fc domain monomer and the fourth polypeptide comprises a sixth Fc domain monomer. In this aspect, a and a 'combine to form a first Fc domain, B and a fifth Fc domain monomer combine to form a second Fc domain, and B' and a sixth Fc domain monomer combine to form a third Fc domain. An exemplary Fc construct of this aspect of the invention is shown in figure 5.
In certain embodiments, a and a' each comprise a dimerization selectivity module that promotes dimerization between the Fc domain monomers. In other embodiments, B and the fifth Fc domain monomers each comprise a dimerization selectivity module that promotes dimerization between the Fc domain monomers. In still other embodiments, B' and the sixth Fc domain monomer each comprise a dimerization selectivity module that promotes dimerization between the Fc domain monomers.
In certain embodiments, one or more of A, B, A ', B', the third polypeptide, and the fourth polypeptide consists of an Fc domain monomer. In one embodiment, A, B, A ', B', the third polypeptide, and the fourth polypeptide are each comprised of an Fc domain monomer.
In certain embodiments, the Fc construct further comprises IgG CLAntibody constant domains and IgG C H1 antibody constant domain, wherein the IgG CLThe antibody constant domains are attached to the IgG C by linkersH1N-terminal of the constant Domain of the antibody, and the IgG C H1 antibody constant domain is attached to the N-terminus of a, e.g. by a linker. In one embodiment, the Fc construct further comprises a second IgG CLAntibody constant domains and second IgG C H1 antibody constant domain, wherein the second IgG CLThe antibody constant domain is attached to the second IgG C, e.g., by a linkerH1 the N-terminal end of the constant domain of the antibody, and the second IgG C H1 antibody constant domains are attached to the N-terminus of a', e.g., by a linker.
In certain embodiments, the Fc construct further comprises a heterologous moiety, such as a peptide, e.g., an albumin binding peptide linked, e.g., via a linker, to the N-terminus or C-terminus of B or B'.
In other embodiments, the first and second polypeptides of the Fc construct have the same amino acid sequence, and the third and fourth polypeptides of the Fc construct have the same amino acid sequence.
In another aspect, the invention relates to an Fc construct comprising two polypeptides. The first polypeptide has the formula a-L-B, wherein a comprises a first Fc domain monomer; l is a linker; and B comprises a serum protein binding moiety, such as albumin binding peptide. The second polypeptide comprises a second Fc domain monomer. In this aspect, the first Fc domain monomer and the second Fc domain monomer combine to form one Fc domain.
In certain embodiments, the first Fc domain monomer and the second Fc domain monomer comprise complementary dimerization selectivity modules that promote dimerization between the first Fc domain monomer and the second Fc domain monomer.
In certain embodiments, a and the second polypeptide are each comprised of an Fc domain monomer.
In yet another aspect, the invention relates to an Fc construct comprising two polypeptides. The first polypeptide has the formula A-L1-B-L2-C, wherein A comprises an IgG CLAn antibody constant domain; l1 and L2 are each a linker; b comprises an IgG C H1 an antibody constant domain; and C comprises a first Fc domain monomer. The second polypeptide has the formula A '-L1' -B '-L2' -C ', wherein A' comprises an IgG CLAn antibody constant domain; l1 'and L2' are each linkers; b' comprises an IgG C H1 an antibody constant domain; and C comprises a second Fc domain monomer. In this aspect, the first Fc domain monomer and the second Fc domain monomer combine to form one Fc domain. An exemplary Fc construct of this aspect of the invention is shown in fig. 7A.
In certain embodiments, the first Fc domain monomer and the second Fc domain monomer comprise dimerization selectivity modules that promote dimerization between the first Fc domain monomer and the second Fc domain monomer.
In certain embodiments, C and C' are each comprised of an Fc domain monomer.
In certain embodiments, the Fc construct further comprises a serum protein binding moiety, such as an albumin binding peptide linked to the N-terminus or C-terminus of C or C' via a linker.
In yet another aspect, the invention relates to a composition comprising four or moreAn Fc construct of a polypeptide. The first polypeptide has the formula A-L1-B-L2-C, wherein A comprises an IgG CLAn antibody constant domain; l1 and L2 are each a linker; b comprises an IgG C H1 an antibody constant domain; and C comprises a first Fc domain monomer. The second polypeptide has the formula A '-L1' -B '-L2' -C ', wherein A' comprises an IgG CLAn antibody constant domain; l1 'and L2' are each linkers; b' comprises an IgG C H1 an antibody constant domain; and C comprises a second Fc domain monomer. In this aspect, the first Fc domain monomer is combined with the third Fc domain monomer to form a first Fc domain, and the second Fc domain monomer is combined with the fourth Fc domain monomer to form a second Fc domain. Furthermore, IgG C of the first polypeptide H1 IgG C of the constant Domain of the antibody and the second polypeptideLCombination of antibody constant domains and IgG C of the second polypeptide H1 IgG C of the constant Domain of the antibody and the first polypeptideLThe antibody constant domains combine to form an Fc construct comprising two or more Fc domains. An exemplary Fc construct of this aspect of the invention is shown in fig. 7B.
In another aspect, the invention relates to an Fc construct comprising two polypeptides. The first polypeptide comprises a first Fc domain monomer and the second polypeptide comprises a second Fc domain monomer. In this aspect, the first Fc domain monomer and the second Fc domain monomer combine to form one Fc domain. An exemplary Fc construct of this aspect of the invention is shown in figure 1. Further, in this aspect, the first Fc domain monomer and the second Fc domain monomer each comprise a dimerization selectivity module that promotes dimerization between the first Fc domain monomer and the second Fc domain monomer. Exemplary Fc constructs of this example are shown in fig. 2 and 3.
In certain embodiments, the first polypeptide and the second polypeptide are each comprised of an Fc domain monomer.
In certain embodiments, the Fc construct further comprises a serum protein binding moiety, e.g., an albumin binding peptide linked to the N-terminus or C-terminus of the first polypeptide or the second polypeptide, e.g., by a linker.
In another aspect, the invention relates to an Fc construct comprising two polypeptides. The first polypeptide has the formula a-L-B, wherein a comprises a first Fc domain monomer; l is a linker; and B comprises a second Fc domain monomer. The second polypeptide has the formula a '-L' -B ', wherein a' comprises a third Fc domain monomer; l' is a linker; and B' comprises a fourth Fc domain monomer. In this aspect, the first Fc domain monomer and the second Fc domain monomer are at their corresponding CH3 antibody constant domains each contain an engineered cavity, and the second and fourth Fc domain monomers are at their respective CH3 an antibody constant domain each comprising an engineered protrusion, wherein the engineered cavity and the engineered protrusion are positioned to form a protrusion-entry-cavity pair. Also in this aspect, the first Fc domain monomer and the third Fc domain monomer combine to form a first Fc domain, and the second Fc domain monomer and the fourth Fc domain monomer combine to form a second Fc domain.
In certain embodiments, one or more of A, B, A 'and B' consists of an Fc domain monomer. In one embodiment, A, B, A 'and B' are each composed of Fc domain monomers.
In certain embodiments, the Fc construct further comprises a serum protein binding moiety, e.g., an albumin binding peptide linked to the N-terminus or C-terminus of B or B', e.g., by a linker.
In certain embodiments, the Fc construct further comprises IgG CLAntibody constant domains and IgG C H1 antibody constant domain, wherein the IgG CLAntibody constant domains are attached to the IgG C, e.g., by a linkerH1N-terminal of the constant Domain of the antibody, and the IgG C H1 antibody constant domain is attached to the N-terminus of a via a linker. In one embodiment, the Fc construct further comprises a second IgG CLAntibody constant domains and second IgG C H1 antibody constant domain, wherein the second IgG CLThe antibody constant domain is attached to the second IgG by a linker C H1 the N-terminal end of the constant domain of the antibody, and the second IgG C H1 antibody constant domains are attached to the N-terminus of a' by a linker.
In another aspect, the invention relates to an Fc construct consisting of: a) a first polypeptide having the formula a-L-B; wherein a comprises or consists of a first Fc domain monomer; l is a linker; and B comprises or consists of a second Fc domain monomer; b) a second polypeptide having the formula a ' -L ' -B '; wherein a' comprises or consists of a third Fc domain monomer; l is a linker; and B' comprises or consists of a fourth Fc domain monomer; c) a third polypeptide comprising or consisting of a fifth Fc domain monomer; and d) a fourth polypeptide comprising or consisting of a sixth Fc domain monomer. A of the first polypeptide and a' of the second polypeptide combine to form a first Fc domain; b of the first polypeptide and the fifth Fc domain monomer combine to form a second Fc domain; and B' of the second polypeptide and the sixth Fc domain monomer combine to form a third Fc domain. Each of the first and third Fc domain monomers comprises a complementary dimerization selectivity module that promotes dimerization between the first and third Fc domain monomers, each of the second and fifth Fc domain monomers comprises a complementary dimerization selectivity module that promotes dimerization between the second and fifth Fc domain monomers, and each of the fourth and sixth Fc domain monomers comprises a complementary dimerization selectivity module that promotes dimerization between the fourth and sixth Fc domain monomers; wherein the Fc construct contains no more than three Fc domains.
In some embodiments of this aspect, one of the first Fc domain monomer or the third Fc domain monomer comprises a negatively charged amino acid substitution and the other Fc domain monomer comprises a positively charged amino acid substitution, the second Fc domain monomer and the fourth Fc domain monomer or one of the fifth Fc domain monomer and the sixth Fc domain monomer comprises an engineered protuberance, and the other Fc domain monomer comprises an engineered cavity. In some embodiments, linker L1, L2, L1 ', and/or L2' is 3-200 amino acids in length. In some embodiments, linker L and/or L' consists of the sequence of any one of SEQ ID NOs 1, 2 and 3.
In another aspect, the invention relates to an Fc construct consisting of: a) a first polypeptide having the formula A-L1-B-L2-C; wherein a comprises or consists of a first Fc domain monomer; l1 is a linker; b comprises or consists of a second Fc domain monomer; l2 is a linker; and C comprises or consists of a third Fc domain monomer; and B) a second polypeptide having the formula A ' -L1 ' -B ' -L2 ' -C '; wherein a' comprises or consists of a fourth Fc domain monomer; l1' is a linker; b' comprises or consists of a fifth Fc domain monomer; l2' is a linker; and C comprises or consists of a sixth Fc domain monomer; c) a third polypeptide comprising or consisting of a seventh Fc domain monomer; d) a fourth polypeptide comprising or consisting of an eighth Fc domain monomer; e) a fifth polypeptide comprising or consisting of a ninth Fc domain monomer; and f) a sixth polypeptide comprising or consisting of a tenth Fc domain monomer. A and a seventh Fc domain monomer of the first polypeptide combine to form a first Fc domain; b of the first polypeptide and B' of the second polypeptide combine to form a second Fc domain; the C and eighth Fc domain monomers of the first polypeptide combine to form a third Fc domain, the a 'and ninth Fc domain monomers of the second polypeptide combine to form a fourth Fc domain, and the C' and tenth Fc domain monomers of the second polypeptide combine to form a fifth Fc domain. Each of the first and seventh Fc domain monomers comprises a complementary dimerization selectivity module that promotes dimerization between the first and seventh Fc domain monomers, each of the second and fifth Fc domain monomers comprises a complementary dimerization selectivity module that promotes dimerization between the second and fifth Fc domain monomers, each of the third and eighth Fc domain monomers comprises a complementary dimerization selectivity module that promotes dimerization between the third and eighth Fc domain monomers; each of the fourth and ninth Fc domain monomers comprises a complementary dimerization selectivity module that promotes dimerization between the fourth Fc domain monomer and the ninth Fc domain monomer; and the sixth and tenth Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the sixth and tenth Fc domain monomers; wherein the Fc construct contains no more than five Fc domains.
In some embodiments of this aspect, the first, third, fourth, and sixth Fc domain monomers each comprise an engineered protrusion, the second Fc domain monomer comprises a negatively charged amino acid substitution, the fifth Fc domain monomer comprises a positively charged amino acid substitution, and the seventh, eighth, ninth, and tenth Fc domain monomers each comprise an engineered cavity. In some embodiments, linker L1, L2, L1 ', and/or L2' is 3-200 amino acids in length. In some embodiments, linker L1, L2, L1 ', and/or L2' consists of the sequence of any one of SEQ ID NOs 1, 2, and 3.
In another aspect, the invention relates to an Fc construct consisting of: a) a first polypeptide having the formula A-L1-B-L2-C; wherein a comprises or consists of a first Fc domain monomer; l1 is a linker; b comprises or consists of a second Fc domain monomer; l2 is a linker; and C comprises or consists of a third Fc domain monomer; and B) a second polypeptide having the formula A ' -L1 ' -B ' -L2 ' -C '; wherein a' comprises or consists of a fourth Fc domain monomer; l1' is a linker; b' comprises or consists of a fifth Fc domain monomer; l2' is a linker; and C comprises or consists of a sixth Fc domain monomer; c) a third polypeptide comprising or consisting of a seventh Fc domain monomer; d) a fourth polypeptide comprising or consisting of an eighth Fc domain monomer; e) a fifth polypeptide comprising or consisting of a ninth Fc domain monomer; f) a sixth polypeptide comprising or consisting of a tenth Fc domain monomer. A of the first polypeptide and a' of the second polypeptide combine to form a first Fc domain; b and a seventh Fc domain monomer of the first polypeptide combine to form a second Fc domain; the C and eighth Fc domain monomers of the first polypeptide combine to form a third Fc domain, the B 'and ninth Fc domain monomers of the second polypeptide combine to form a fourth Fc domain, and the C' and tenth Fc domain monomers of the second polypeptide combine to form a fifth Fc domain. Each of the first and fourth Fc domain monomers comprises a complementary dimerization selectivity module that promotes dimerization between the first and fourth Fc domain monomers, each of the second and seventh Fc domain monomers comprises a complementary dimerization selectivity module that promotes dimerization between the second and seventh Fc domain monomers, each of the third and eighth Fc domain monomers comprises a complementary dimerization selectivity module that promotes dimerization between the third and eighth Fc domain monomers; each of the fifth and ninth Fc domain monomers comprises a complementary dimerization selectivity module that promotes dimerization between the fifth Fc domain monomer and the ninth Fc domain monomer; and the sixth and tenth Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the sixth and tenth Fc domain monomers; wherein the Fc construct contains no more than five Fc domains.
In some embodiments of this aspect, the first Fc domain monomer comprises a negatively charged amino acid substitution, the fourth Fc domain monomer comprises a positively charged amino acid substitution, the second, third, fifth, and sixth Fc domain monomers each comprise an engineered protuberance, and the seventh, eighth, ninth, and tenth Fc domain monomers each comprise an engineered cavity. In some embodiments, linker L1, L2, L1 ', and/or L2' is 3-200 amino acids in length. In some embodiments, linker L1, L2, L1 ', and/or L2' consists of the sequence of any one of SEQ ID NOs 1, 2, and 3.
In another aspect, the invention relates to an Fc construct comprising one or more Fc domains, wherein the Fc construct is assembled from a single polypeptide sequence. The polypeptide has the formula a-L-B, wherein a comprises a first Fc domain monomer; l is a linker (optionally a cleavable linker with, for example, one, two, or more cleavage sites); and B comprises a second Fc domain monomer. The linker can be an amino acid spacer of sufficient length (e.g., at least 15 amino acids, preferably at least about 20 amino acid residues in length, such as 15-200 amino acids in length) and flexibility (the first and second Fc domain monomers of the polypeptide combine to form an Fc domain). In certain embodiments, the first Fc domain monomer and the second Fc domain monomer comprise complementary dimerization selectivity modules that promote dimerization between the first Fc domain monomer and the second Fc domain monomer. Such a construct may be formed by expression of a single polypeptide sequence in a host cell. In one embodiment, the polypeptide has the formula a-L1-B-L2-C, wherein a comprises a first Fc domain monomer; l1 is a linker (optionally a cleavable linker with, for example, one, two or more cleavage sites); b comprises a second Fc domain monomer; l2 is a linker; and C is a third Fc domain monomer. The linker can be an amino acid spacer of sufficient length (e.g., at least 15 amino acids, preferably at least about 20 amino acid residues in length, such as 15-200 amino acids in length) and flexibility (the first and second Fc domain monomers of the polypeptide combine to form an Fc domain). In certain embodiments, the first Fc domain monomer and the second Fc domain monomer comprise complementary dimerization selectivity modules that promote dimerization between the first Fc domain monomer and the second Fc domain monomer. An example of the Fc construct of this embodiment, which comprises three Fc domains, is depicted in figure 10.
In any of the Fc constructs described herein, the Fc domain monomers of the Fc domains of the constructs may have the same primary amino acid sequence. For example, both Fc domain monomers of an Fc domain can be wild-type sequences, or both Fc domain monomers of an Fc domain can have the same dimerization selectivity module, e.g., both Fc domain monomers of an Fc domain can be at CH3 domain at the interface between the ring of charged residues at least two positions with the same opposite charge mutation.
In any of the Fc constructs described herein, the Fc domain monomers of the Fc domain of the construct may have different sequences, e.g., sequences that differ by no more than 20 amino acids (e.g., no more than 15, 10 amino acids), e.g., no more than 20, 15, 10, 8, 7, 6, 5, 4, 3, or 2 amino acids, between two Fc monomers (i.e., between the Fc domain monomer of the Fc construct and another monomer). For example, the Fc monomer sequences of the constructs described herein may be different, as the complementary dimerization selectivity module of any Fc construct may comprise C in one domain monomerH3 one engineered cavity in the constant domain of the antibody and C in another Fc domain monomerH3 an engineered protuberance in an antibody constant domain, wherein the engineered cavity and the engineered protuberance are positioned to form a protuberance-into-cavity pair of Fc domain monomers. Exemplary engineered cavities and protrusions are shown in table 1. In other embodiments, the complementary dimerization selectivity module comprises C in one domain monomerH3 one engineered (substituted) negatively charged amino acid in the constant domain of the antibody and C in the other Fc domain monomerH3 an engineered (substituted) positively charged amino acid in the constant domain of an antibody, wherein the negatively charged amino acid and the positively charged amino groupThe acid is positioned to facilitate formation of the Fc domain between the complementary domain monomers. Exemplary complementary amino acid changes are shown in table 2.
In some embodiments, in addition to dimerization selectivity modules (e.g., engineered cavities and protrusions, or engineered positively and negatively charged amino acids (see, e.g., exemplary amino acid changes in tables 1 and 2)), the Fc constructs described herein may further comprise additional amino acid substitutions in the Fc monomer sequence from the wild-type sequence, e.g., to help stabilize the Fc construct or prevent protein aggregation.
In some embodiments, an Fc construct described herein comprises 2-10 Fc domains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 domains), wherein at least two of the Fc domains of the construct have different dimerization selectivity modules. For example, constructs 5, 8, 9, and 10 have at least one Fc domain comprising an engineered cavity and overhang and at least one Fc domain comprising a complementary opposite charge mutation.
In other embodiments, one or more linkers in the Fc constructs described herein is a bond.
In other embodiments, one or more linkers in the Fc constructs described herein is a spacer, e.g., an amino acid spacer having 2-200 amino acids.
In certain embodiments, the amino acid spacer is a glycine-and/or serine-rich spacer, e.g., the spacer comprises two or more motifs of the sequences GS, GGS, GGGGS (SEQ ID NO:1), GGSG (SEQ ID NO:2), or SGGG (SEQ ID NO: 3).
In certain embodiments, when the Fc construct comprises an albumin binding peptide, the albumin binding peptide has the sequence of DICLPRWGCLW (SEQ ID NO: 28).
In other embodiments, one or more Fc domain monomers in the Fc constructs described herein comprise an IgG hinge domain, an IgG C H2 antibody constant Domain, and IgG CH3 an antibody constant domain.
In certain embodiments, of the foregoing Fc constructsEach Fc domain monomer comprises an IgG hinge domain, IgG C H2 antibody constant Domain, and IgG CH3 an antibody constant domain.
In certain embodiments, the IgG has a subtype selected from the group consisting of: IgG1, IgG2a, IgG2b, IgG3, and IgG 4.
In yet another aspect, the invention relates to a pharmaceutical composition comprising a substantially homologous (e.g., at least 85%, 90%, 95%, 97%, 98%, 99% homologous) collection of any of the Fc constructs described herein. In one embodiment, a sterile syringe or vial that is acceptable for pharmaceutical use contains a pharmaceutical composition in which the sole or primary active ingredient is a substantially homologous (e.g., at least 85%, 90%, 95%, 98%, or 99% homologous) collection of any one of the Fc constructs described herein. The pharmaceutical composition may comprise one or more inactive ingredients, e.g. selected from salts, detergents, surfactants, bulking agents, polymers, preservatives, and other pharmaceutically acceptable excipients, in another embodiment the substantially homogeneous pharmaceutical composition contains less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% aggregates or unwanted multimers of the Fc construct.
In another aspect, the invention relates to a method of making any one of the aforementioned Fc constructs. The method comprises providing a host cell comprising one or more polynucleotides encoding polypeptides required to assemble the Fc construct, expressing the polypeptides in the host cell under conditions that allow formation of the Fc construct, and recovering (e.g., purifying) the Fc construct.
In some embodiments, the Fc construct is formed, at least in part, by association of Fc domain monomers present in different polypeptides. In certain embodiments, the Fc construct is formed by association of Fc domain monomers present in different polypeptides. In these embodiments, the Fc construct does not comprise an additional domain (e.g., an IgM tail or an IgA tail) that facilitates association of the two polypeptides. In other embodiments, a covalent bond (e.g., a disulfide bond) is present only between two Fc domain monomers that are linked to form an Fc domain. In other embodiments, the Fc construct does not comprise covalent bonds (e.g., disulfide bonds) between Fc domains. In still other embodiments, the Fc construct provides sufficient structural flexibility such that all or substantially all of the Fc domains in the Fc construct are capable of interacting with Fc receptors on the surface of a cell simultaneously. In certain instances of any of these embodiments, the Fc construct comprises at least two Fc domains connected by a linker (e.g., a flexible amino acid spacer).
In one embodiment, the Fc domain monomers of the Fc domain are found in different polypeptide chains that associate to form the Fc domain. For example, the constructs depicted in fig. 4 and 6 have two Fc domains comprising three associated polypeptides. One of the three polypeptides comprises two Fc domain monomers and the other two of the polypeptides each comprise one Fc domain monomer. The construct depicted in figure 5 has three Fc domains comprising four associated polypeptides; two of the four polypeptides have two Fc domain monomers and the other two of the four polypeptides each have one Fc domain monomer. The Fc construct depicted in fig. 7B may have n Fc domains comprising 2n polypeptides (where n is 2-10), each polypeptide comprising one Fc domain monomer, one IgG CLAntibody constant domains, and an IgG C H1 antibody constant domain. The constructs depicted in fig. 8 and 9 each have five Fc domains comprising six associated polypeptides. Two of the six polypeptides have three Fc domain monomers and the other four of the six polypeptides each have one Fc domain monomer. The construct depicted in figure 10 has three Fc domains comprising two associated polypeptides. Each of these two polypeptides contains three Fc domain monomers connected in tandem.
In another aspect, the invention relates to compositions and methods for promoting selective dimerization of Fc domain monomers. The invention includes an Fc domain, wherein two Fc domain monomers of the Fc domain are at CH3 at least two positions within the ring of charged residues at the interface between the constant domains of the antibodyThe same mutation. The invention also includes a method of making such an Fc domain comprising introducing a monomer sequence at C in both Fc domainsH3 complementary dimerization selectivity module having the same mutations at least two positions within a loop of charged residues at the interface between antibody constant domains. At CH3 the interface between the antibody constant domains consists of a hydrophobic sheet (patch) surrounded by a charged residue ring. When a CH3 antibody constant domains are bound together, these charged residues pair with residues of opposite charge. By reversing the charge of both members of two or more complementary residue pairs, the mutated Fc domain monomer remains complementary to an Fc domain monomer of the same mutated sequence, but has a lower complementarity to an Fc domain monomer that does not have these mutations. In this embodiment, the same dimerization selectivity module promotes homodimerization. Exemplary Fc domains include Fc monomers comprising double mutants K409D/D399K, K392D/D399K, E357K/K370E, D356K/K439D, K409E/D399K, K392E/D399K, E357K/K370D, or D356K/K439E. In another embodiment, the Fc domain comprises Fc monomers including four mutants in combination with any double mutant pair, e.g., K409D/D399K/E357K/K370E. In another embodiment, in addition to identical dimerization selectivity modules, the Fc domain monomers of the Fc domain comprise complementary dimerization selectivity modules (e.g., engineered cavities and protrusions) with different mutations that promote specific associations. As a result, the two Fc domain monomers comprise two dimerization selectivity modules and remain complementary to each other, but have reduced complementarity to the other Fc domain monomers. This example promotes heterodimerization between cavity-containing Fc domain and bulge-containing Fc domain monomers. In one example, the same mutations in the charged pair residues of two Fc domain monomers are combined with protrusions on one Fc domain monomer and cavities on the other Fc domain monomer.
In another aspect, the invention relates to a method of reducing inflammation in a subject in need thereof. In another aspect, the invention relates to a method of promoting clearance of autoantibodies in a subject in need thereof. In another aspect, the invention relates to a method of inhibiting antigen presentation in a subject in need thereof. In another aspect, the invention relates to a method of reducing an immune response in a subject in need thereof, e.g., a method of reducing immune response in a subject in need thereof based on activation of an immune complex. The methods comprise administering to the subject an Fc construct or pharmaceutical composition described herein.
In another aspect, the invention relates to a method of treating inflammation or an autoimmune or immune disease in a subject in need thereof by administering to the subject an Fc construct or pharmaceutical composition described herein (e.g., any one of constructs 1-10 and 5). Exemplary diseases include: rheumatoid Arthritis (RA); systemic Lupus Erythematosus (SLE); ANCA-associated vasculitis; antiphospholipid antibody syndrome; autoimmune hemolytic anemia; chronic inflammatory demyelinating neuropathies; clearance of anti-allogenic in transplants, anti-autoantibodies in GVHD, anti-surrogate, IgG therapeutic, IgG aberrant proteins; dermatomyositis; goodpasture's syndrome; targeted organ system type II hypersensitivity syndromes mediated by antibody-dependent cell-mediated cytotoxicity, such as gilland barre syndrome, CIDP, dermatomyositis, feldian syndrome, antibody-mediated rejection, autoimmune thyroid disease, ulcerative colitis, autoimmune liver disease; idiopathic thrombocytopenic purpura; myasthenia gravis, neuromyelitis optica; pemphigus and other autoimmune foaming disorders; scleroderma; autoimmune cytopenia and other disorders mediated by antibody-dependent phagocytosis; other FcR-dependent inflammatory syndromes such as synovitis, dermatomyositis, systemic vasculitis, glomerulonephritis and vasculitis.
In another aspect, the invention relates to an Fc construct or pharmaceutical composition described herein (e.g., any one of constructs 1-10 and 5) for use in reducing inflammation in a subject in need thereof. In another aspect, the invention relates to an Fc construct or pharmaceutical composition described herein (e.g., any one of constructs 1-10 and 5) for use in promoting autoantibody clearance in a subject in need thereof. In another aspect, the invention relates to an Fc construct or pharmaceutical composition described herein (e.g., any one of constructs 1-10 and 5) for use in inhibiting antigen presentation in a subject in need thereof. In another aspect, the invention relates to an Fc construct or pharmaceutical composition described herein (e.g., any one of constructs 1-10 and 5) for use in reducing an immune response in a subject in need thereof, e.g., reducing an immune response in a subject in need thereof based on activation of an immune complex.
In another aspect, the invention relates to an Fc construct or pharmaceutical composition described herein (e.g., any one of constructs 1-10 and 5) for use in treating an inflammatory or autoimmune or immune disease in a subject. Exemplary diseases include: rheumatoid Arthritis (RA); systemic Lupus Erythematosus (SLE); ANCA-associated vasculitis; antiphospholipid antibody syndrome; autoimmune hemolytic anemia; chronic inflammatory demyelinating neuropathies; clearance of anti-allogenic in transplants, anti-autoantibodies in GVHD, anti-surrogate, IgG therapeutic, IgG aberrant proteins; dermatomyositis; goodpasture's syndrome; targeted organ system type II hypersensitivity syndromes mediated by antibody-dependent cell-mediated cytotoxicity, such as gilland barre syndrome, CIDP, dermatomyositis, feldian syndrome, antibody-mediated rejection, autoimmune thyroid disease, ulcerative colitis, autoimmune liver disease; idiopathic thrombocytopenic purpura; myasthenia gravis, neuromyelitis optica; pemphigus and other autoimmune foaming disorders; scleroderma; autoimmune cytopenia and other disorders mediated by antibody-dependent phagocytosis; other FcR-dependent inflammatory syndromes such as synovitis, dermatomyositis, systemic vasculitis, glomerulonephritis and vasculitis.
In any of the Fc constructs described herein, it is understood that the order of the Fc domain monomers is interchangeable. For example, in a polypeptide of formula a-L-B, the carboxy terminus of a may be linked to the amino terminus of L, which in turn is linked at its carboxy terminus to the amino terminus of B. Alternatively, the carboxy terminus of B may be linked to the amino terminus of L, which in turn is linked at its carboxy terminus to the amino terminus of C. Both of these configurations are encompassed by the formula A-L-B.
In a related aspect, the invention relates to a host cell expressing any one of the aforementioned Fc constructs. The host cell comprises polynucleotides encoding polypeptides required for assembly of the Fc construct, wherein the polynucleotides are expressed in the host cell.
Defining:
as used herein, the term "Fc domain monomer" refers to a monomer comprising at least one hinge domain and second and third antibody constant domains (C)H2 and CH3) Or a functional fragment thereof (e.g., a fragment that is capable of (i) dimerizing with another Fc domain monomer to form an Fc domain and (ii) binding an Fc receptor). The Fc domain monomer can be any immunoglobulin antibody isotype including IgG, IgE, IgM, IgA, or IgD. In addition, the Fc domain monomer may be of the IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, and IgG 4). The Fc domain monomer does not comprise any immunoglobulin portion capable of acting as an antigen recognition region (e.g., a variable domain or Complementarity Determining Region (CDR)). The Fc domain monomer may contain up to ten changes (e.g., 1-10, 1-8, 1-6, 1-4 amino acid substitutions, additions, or deletions) from the wild-type Fc domain monomer sequence that alter the interaction between the Fc domain and the Fc receptor. Examples of suitable variations are known in the art.
As used herein, the term "Fc domain" refers to a dimer of two Fc domain monomers capable of binding to an Fc receptor. In the wild-type Fc domain, the two Fc domain monomers pass through two CsH3, and one or more disulfide bonds formed between the hinge domains of the two dimerizing Fc domain monomers.
In the present invention, the term "Fc construct" refers to an associated polypeptide chain formed between 2-10 Fc domains as described herein. The Fc constructs described herein may comprise Fc domain monomers having the same or different sequences. For example, Fc constructsThere may be two Fc domains, one of which comprises an IgG1 or IgG1 derived Fc domain monomer and the second of which comprises an IgG2 or IgG2 derived Fc domain monomer. In another example, an Fc construct may have two Fc domains, one of which comprises a "protuberance-entry-cavity pair" and the second does not. In the present invention, the Fc domain does not comprise the variable region of an antibody, e.g., VH、VLCDR, or HVR. The Fc domain forms the minimal structure that binds to Fc receptors such as Fc γ RI, Fc γ RIIa, Fc γ RIIb, Fc γ RIIIa, Fc γ RIIIb, Fc γ RIV.
As used herein, the term "antibody constant domain" refers to a constant region domain (e.g., C) corresponding to an antibodyLAntibody constant domains, C H1 antibody constant Domain, C H2 antibody constant Domain, or CH3 antibody constant domain).
As used herein, the term "promoting" is intended to encourage and promote, for example, the formation of an Fc domain from two Fc domain monomers that have a higher binding affinity for each other than for other different Fc domain monomers. As described herein, two Fc domain monomers that combine to form an Fc domain are at their corresponding CH3 antibody constant domain interfaces can have compatible amino acid modifications (e.g., engineered protrusions and engineered cavities). Compatible amino acid modifications facilitate or contribute to selective interaction of such Fc domain monomers with each other relative to other Fc domain monomers lacking such amino acid modifications or having incompatible amino acid modifications. This is because of the presence of C at two interacting sitesH3 antibody constant domain, the Fc domain monomers have a higher affinity for each other than for other Fc domain monomers lacking the amino acid modifications.
As used herein, the term "dimerization selectivity module" refers to a sequence of Fc domain monomers that facilitates preferential pairing between two Fc domain monomers. A "complementary" dimerization selectivity module is a dimerization selectivity module that promotes or contributes to the selective interaction of two Fc domain monomers with each other. The complementary dimerization selectivity modules may have the same or different sequences. Exemplary complementary dimerization selectivity modules are described herein.
As used herein, the term "engineered cavity" refers to the replacement of C with a different amino acid residue having a smaller side chain volume than the original amino acid residueH3 at least one original amino acid residue in the constant domain of the antibody, thereby being at CH3 a three-dimensional cavity is formed in the antibody constant domain. The term "original amino acid residue" refers to a residue consisting of wild type CH3 the naturally occurring amino acid residues encoded by the genetic code of the constant domain of the antibody.
As used herein, the term "engineered overhang" refers to the replacement of C with a different amino acid residue having a side chain volume greater than the original amino acid residueH3 at least one original amino acid residue in the constant domain of the antibody, thereby being at CH3A three-dimensional protrusion is formed in the constant domain of the antibody. The term "original amino acid residue" refers to a residue derived from wild-type CH3 the naturally occurring amino acid residues encoded by the genetic code of the constant domain of the antibody.
As used herein, the term "protuberance-into-cavity pair" describes an Fc domain comprising two Fc domain monomers, wherein the first Fc domain monomer is at its CH3 antibody constant Domain comprising an engineered Cavity, and a second Fc Domain monomer at its CH3 antibody constant domains contain an engineered overhang. C of first Fc domain monomer in one protrusion-entry-cavity pairH3 engineered overhang in antibody constant domain positioned such that it is aligned with the C of the second Fc domain monomerH3 engineered cavity interactions in antibody constant domains without significant interference at CH3 normal association of dimers at the interface between constant domains of the antibody.
As used herein, the term "linked" is used to describe two or more elements, components, or protein domains (e.g., polypeptides) by including chemical couplings, recombinant means, and chemical bonds (e.g., two)Sulfide and amide bonds) or attachment. For example, two individual polypeptides may be linked by chemical coupling, chemical linkage, peptide linker, or any other means of covalent linkage to form one contiguous protein structure. In some embodiments, the first Fc domain monomer is linked to the second Fc domain monomer by a peptide linker, wherein the N-terminus of the peptide linker is linked to the C-terminus of the first Fc domain monomer by a chemical bond (e.g., a peptide bond), and the C-terminus of the peptide linker is linked to the N-terminus of the second Fc domain monomer by a chemical bond (e.g., a peptide bond). In other embodiments, the N-terminus of the albumin binding peptide is linked to the C of the Fc domain monomer by a linker in the same manner as described aboveH3 the C-terminus of the constant domain of the antibody.
As used herein, the term "associate" is used to describe interactions (e.g., hydrogen bonding, hydrophobic interactions, or ionic interactions) between polypeptides (or sequences within a single polypeptide) such that the polypeptides (or sequences within a single polypeptide) are positioned to form an Fc construct having at least one Fc domain. For example, two polypeptides each comprising one Fc domain monomer can associate to form an Fc construct (e.g., as depicted in fig. 1-3). In some embodiments, three polypeptides, e.g., one polypeptide comprising two Fc domain monomers and two polypeptides each comprising one Fc domain monomer, associate to form an Fc construct having two Fc domains (e.g., as shown in fig. 4 and 6). In some embodiments, four polypeptides, e.g., two polypeptides each comprising two Fc domain monomers and two polypeptides each comprising one Fc domain monomer, associate to form an Fc construct having three Fc domains (e.g., as depicted in fig. 5). In other embodiments, 2n polypeptides, e.g., comprising an Fc domain monomer, IgG CLAntibody constant domain, and IgG C H1 to form an Fc construct with n Fc domains (as depicted in figure 7B). The two polypeptides may be associated by their respective Fc domain monomers, or by other components of the polypeptide. For example, in FIG. 7B, polypeptide 708 is conjugated to a polypeptide via its Fc domain monomerPeptide 706 associates and through its CLDomain and C of polypeptide 710H1 domain association to associate with polypeptide 710. The association between polypeptides does not comprise covalent interactions. For example, in figure 10, Fc monomer sequences 1014 and 1012 within a single polypeptide associate to form an Fc domain, as do Fc monomer sequences 1004 and 1006.
As used herein, the term "linker" refers to a connection between two elements (e.g., protein domains). The linker may be a covalent bond or a spacer. The term "bond" refers to a chemical bond (e.g., an amide bond or a disulfide bond), or any type of bond formed by a chemical reaction, such as a chemical coupling. The term "spacer" refers to a moiety (e.g., a polyethylene glycol (PEG) polymer) or an amino acid sequence (e.g., 3-200 amino acids, 3-150 amino acids, or 3-100 amino acid sequences) that is present between two polypeptide or polypeptide domains in order to provide space and/or flexibility between the two polypeptide or polypeptide domains. An amino acid spacer is a portion of the primary sequence of a polypeptide (e.g., a polypeptide or polypeptide domain linked to the spacer via a polypeptide backbone). For example, disulfide bonds formed between two hinge regions or two Fc domain monomers forming an Fc domain are not considered to be linkers.
As used herein, the term "cleavable linker" refers to a linker containing one or more elements that can be selectively cleaved, e.g., after formation of the construct, e.g., the cleavable linker includes a polypeptide sequence that can be selectively cleaved by a protease.
As used herein, the term "albumin binding peptide" refers to an amino acid sequence of 12 to 16 amino acids that has an affinity for and functions to bind serum albumin. Albumin binding peptides may be of different origin, e.g. human, mouse, or rat. In some embodiments of the invention, the albumin binding peptide is fused to the C-terminus of the Fc domain monomer to increase the serum half-life of the Fc construct. The albumin binding peptide may be fused to the N-terminus or C-terminus of the Fc domain monomer, either directly or through a linker.
As used herein, the term "multimer" refers to a molecule comprising at least two associated Fc constructs described herein.
As used herein, the term "polynucleotide" refers to an oligonucleotide, or nucleotide, and fragments or portions thereof, and refers to DNA or RNA of genomic or synthetic origin that may be single-stranded or double-stranded and represents the sense or antisense strand. A single polynucleotide is translated into a single polypeptide.
As used herein, the term "polypeptide" describes a single polymer in which the monomers are amino acid residues bonded together through amide bonds. A polypeptide is intended to include any amino acid sequence, naturally occurring, recombinant, or synthetically produced.
As used herein, the term "amino acid position" refers to the position number of an amino acid in a protein or protein domain. The amino acid positions of the antibodies or Fc constructs were numbered using the Kabat numbering system (Kabat numbering system) (Kabat et al, Sequences of proteins of immunological Interest, National Institutes of Health, Bethesda, Md., 5 th edition, 1991, National Institutes of Health, bessesda, maryland, and the like).
As used herein, the term "host cell" refers to a vector that contains the necessary cellular components (e.g., organelles) required for expression of a protein from its corresponding nucleic acid. These nucleic acids are typically contained in nucleic acid vectors, which can be introduced into host cells by conventional techniques known in the art (transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). The host cell can be a prokaryotic cell, such as a bacterial cell, or a eukaryotic cell, such as a mammalian cell (e.g., a CHO cell). As described herein, the host cell is used to express one or more polypeptides encoding the desired domains, which can then be combined to form the desired Fc construct.
As used herein, the term "pharmaceutical composition" refers to a pharmaceutical or pharmaceutical formulation containing an active ingredient and one or more excipients and diluents such that the active ingredient is suitable for use in a method of administration. The pharmaceutical compositions of the invention comprise a pharmaceutically acceptable component compatible with the Fc construct. The pharmaceutical compositions are typically in the form of aqueous solutions for intravenous or subcutaneous administration.
As used herein, a "substantially homologous collection" of polypeptides or Fc constructs is a collection in which at least 85% of the polypeptides or Fc constructs in a composition (e.g., a pharmaceutical composition) have the same number of Fc domains and the same Fc domain structure. In various embodiments, at least 90%, 92%, 95%, 97%, 98%, 99%, or 99.5% of the polypeptides or Fc constructs in the composition are identical. Thus, a pharmaceutical composition comprising a substantially homologous collection of Fc constructs is one in which at least 85% of the Fc constructs in the composition have the same number of Fc domains and the same structure. A substantially homologous collection of Fc constructs does not include more than 10% (e.g., no more than 8%, 5%, 2%, or 1%) of multimers or aggregates of the Fc construct.
As used herein, the term "pharmaceutically acceptable carrier" refers to an excipient or diluent in a pharmaceutical composition. The pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not deleterious to the recipient. In the present invention, a pharmaceutically acceptable carrier must provide adequate drug stability to the Fc construct. The nature of the carrier will vary depending on the mode of administration. For example, for oral administration, solid carriers are preferred; for intravenous administration, aqueous carriers (e.g., WFI and/or buffered solutions) are typically used.
As used herein, "therapeutically effective amount" refers to an amount, e.g., a pharmaceutical dose, effective to induce a desired biological effect in a subject or patient or to treat a patient having a condition or disorder described herein. It is also understood herein that a "therapeutically effective amount" may be interpreted as an amount that produces the desired therapeutic effect, either alone or in combination with other therapeutic agents, in one dose or in any dose or route.
Drawings
Figure 1 is a schematic representation of an Fc construct (construct 1) containing a dimer of two wild-type (wt) Fc domain monomers (102 and 104).
Figure 2 is a schematic representation of an Fc construct containing a dimer of two Fc domain monomers (construct 2). First Fc domain monomer (202) at C thereofH3 antibody constant Domain contains a protuberance and the second Fc Domain monomer (204) is at its CH3A cavity is contained in the juxtaposed position in the antibody constant domain.
Figure 3 is a schematic representation of another Fc construct (construct 3). This Fc construct contains a dimer of two Fc domain monomers (302 and 304) in which the two Fc domain monomers are at their CH3-CHEach 3 interface contains a charged amino acid different from the wt sequence to promote favorable electrostatic interactions between the two Fc domain monomers.
Figure 4 is a schematic representation of an Fc construct containing two Fc domains (construct 4). This construct is formed from three polypeptides. The first polypeptide (402) contains two wt Fc domain monomers (404 and 406) connected in tandem. The second and third polypeptides (408 and 410, respectively) each contain a wt Fc domain monomer.
Figure 5 is a schematic representation of an Fc construct (construct 5 or construct 5) containing three Fc domains formed by four polypeptides. The first polypeptide (502) is contained at CH3-CH3 an Fc domain monomer (506) having a charged amino acid at the interface different from the wt sequence, the Fc domain monomer linked in tandem to the protuberance-containing Fc domain monomer (504). The second polypeptide (508) is contained at CH3-CH3 having a charged amino acid different from the wt sequence, and is linked in tandem to another protuberance-containing Fc domain monomer (510). The third and fourth polypeptides (514 and 516, respectively) each contain a cavity-containing Fc domain monomer.
Figure 6 is a schematic representation of an Fc construct (construct 6) containing two Fc domains formed by three polypeptides. The first polypeptide (602) contains two knob-containing Fc domain monomers (604 and 606) connected in tandem, while the second and third polypeptides (608 and 610, respectively) each contain one Fc domain monomer engineered to contain a corresponding cavity.
Figure 7A is a schematic representation of another Fc construct (construct 7). The Fc construct contains two CL-CHDimers of 1-Fc domain monomers (702 and 704). In this example, CLThe antibody constant domains have been linked to adjacent C H1 antibody constant domain.
FIG. 7B is a C with multiple Fc domainsL-CHSchematic representation of an Fc construct (construct 8) of multimers of 1-Fc domain monomers (e.g., 706, 708, and 710). In this Fc construct, the constitutive polypeptide may be the same as the constitutive polypeptide in construct 7. C of one Fc constructLC of antibody constant Domain (e.g., 712) with second Adjacent Fc constructH1 antibody constant domains (e.g., 714).
Figure 8 is a schematic representation of an Fc construct (construct 9) containing five Fc domains formed from six polypeptides. The first and second polypeptides (802 and 810) each contain three Fc domain monomers (804, 806, 808 and 812, 814, 816, respectively) connected in tandem. Specifically, in polypeptide 802 or 810, a first overhang-containing Fc domain monomer (804 or 812) is linked at CH3-CHA second Fc domain monomer (806 or 814) containing a charged amino acid at the 3 interface that is different from the wt sequence, the second Fc domain monomer being linked to a third knob-containing Fc domain monomer (808 or 816). The third through sixth polypeptides (818, 820, 822, and 824) each contain one cavity-containing Fc domain monomer and form Fc domains with each Fc domain monomer 804, 808, 812, and 816, respectively.
Figure 9 is a schematic representation of an Fc construct (construct 10) containing five Fc domains formed from six polypeptides. The first and second polypeptides (902 and 910) each contain three Fc domain monomers (904, 906, 908 and 912, 914, 916, respectively) connected in tandem. Specifically, in polypeptide 902 or 910, a first overhang-containing Fc domain monomer (904 or 912) is linked to a second overhang-containing Fc domain monomer (906 or 914) linked at CH3-CH3 at the interface containst different charged amino acids (908 or 916). The third through sixth polypeptides (918, 920, 922, and 924) each contain one cavity-containing Fc domain monomer and form an Fc domain with each Fc domain monomer 904, 906, 912, and 914, respectively.
Figure 10 is a schematic representation of an Fc construct (construct 11) containing three Fc domains formed from two polypeptides having the same sequence. Each of these two polypeptides (1002 and 1010) contains three Fc domain monomers (1004, 1006, 1008 and 1012, 1014, 1016, respectively) connected in tandem. Specifically, each polypeptide contains a first protuberance-containing Fc domain monomer (1004 or 1012) linked to a second cavity-containing Fc domain monomer (1006 or 1014) linked at CH3-CH3 a third Fc domain monomer (1008 or 1016) having a charged amino acid at the interface that is different from the wt sequence. Fc domain monomers 1008 and 1016 associate to form a first Fc domain; fc domain monomers 1004 and 1006 associate to form a second Fc domain; and Fc domain monomers 1012 and 1014 associate to form a third Fc domain. Construct 11 may be formed by expression of a single polypeptide sequence in a host cell.
FIGS. 11A-11B show reducing and non-reducing SDS-PAGE, respectively, for construct 4.
FIGS. 12A-12B show reducing and non-reducing SDS-PAGE, respectively, for construct 6.
Figure 13 is SDS-PAGE of construct 5 and a table showing the percentage of protein expressed before and after purification of construct 5, having three Fc domains (trimers), two Fc domains (dimers), or one Fc domain (monomers).
Fig. 14A and 14B show THP-1 monocyte activation (fig. 14A) and blocking (fig. 14B) assays using constructs 1, 5, and 6.
FIG. 15 shows the effect of IVIG and constructs 5 and 6 in the K/BxN model of rheumatoid arthritis.
Figure 16 shows the effect of IVIG and constructs 5 and 6 in the chronic ITP model.
FIG. 17 shows the inhibition of phagocytosis in THP-1 monocytes by IVIg or construct 5.
Detailed description of the invention
Therapeutic proteins comprising the Fc domain of IgG can be used to treat inflammation as well as immunological and inflammatory diseases. The present invention relates to compositions and methods for making different Fc constructs containing two or more (e.g., 2-10) Fc domains.
Fc domain monomers
The Fc domain monomer comprises a hinge domain, a C H2 antibody constant Domain and a CH3 an antibody constant domain. The Fc domain monomer may be of the immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. The Fc domain monomer can also be any immunoglobulin antibody isotype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG 4). A dimer of Fc domain monomers is an Fc domain (further defined herein) that can bind to an Fc receptor (e.g., fcyriiia, which is a receptor located on the surface of leukocytes). In the present invention, C of Fc domain monomerH3 the antibody constant domains may be at CH3-CH3 antibody constant domains contain amino acid substitutions at the interface to facilitate their association with each other. In some embodiments, the Fc domain monomer comprises two other constant domains, e.g., C, attached to the N-terminusLAntibody constant domains and C H1 antibody constant domain (figure 7). In other embodiments, the Fc domain monomer comprises one additional moiety, such as an albumin binding peptide, attached to the C-terminus. In the present invention, the Fc domain monomer does not contain any type of antibody variable region, e.g., VH、VLComplementarity Determining (CDR), or hypervariable region (HVR).
Fc domains
As defined herein, an Fc domain comprises a through CH3 interaction between antibody constant domains two Fc domain monomers that dimerize. In the present invention, the Fc domain does not comprise the variable region of an antibody, e.g., VH、VLCDR, or HVR. The Fc domain forms the minimal structure that binds to the Fc receptorFor example, Fc γ RI, Fc γ RIIa, Fc γ RIIb, Fc γ RIIIa, Fc γ RIIIb, Fc γ RIV.
Dimerization selectivity Module
In the present invention, the dimerization selectivity module is part of an Fc domain monomer that facilitates the preferential pairing of two Fc domain monomers to form an Fc domain. Specifically, the dimerization selectivity module is C of an Fc domain monomerH3 antibody constant domains comprise a C positioned at the interaction of two Fc monomersH3 a portion of an amino acid substitution at the interface between antibody constant domains. In the dimerization selectivity module, these amino acid substitutions result in two CsH3 antibody constant domains are favored for dimerization due to the compatibility of the amino acids selected for these substitutions. The Fc domain may be selected to ultimately form a favorable Fc domain relative to other Fc domains formed from Fc domain monomers lacking the dimerization selectivity module or having incompatible amino acid substitutions in the dimerization selectivity module. Amino acid substitutions of this type can be made using conventional molecular cloning techniques well known in the art, such as
Figure BDA0003149346320000191
And (4) carrying out mutagenesis.
In some embodiments, the dimerization selectivity module is contained in CH3 engineered cavities in the antibody constant domains (described further herein). In other embodiments, the dimerization selectivity module is contained in CH3 engineered protrusions in the constant domain of the antibody (described further herein). To selectively form an Fc domain, two Fc domain monomers (e.g., one C containing an engineered cavity) with compatible dimerization selectivity modules are combinedH3 antibody constant Domain and another C comprising an engineered overhangH3 antibody constant domains) to form a protuberance-entry-cavity pair of Fc domain monomers.
In other embodiments, an Fc domain monomer having a dimerization selectivity module with a positively charged amino acid substitution and an Fc domain monomer having a dimerization selectivity module with a negatively charged amino acid substitution may be selectively combined to form an Fc domain through favorable electrostatic steering of charged amino acids (described further herein). Specific dimerization selectivity modules are further listed in table 1 and table 2 (but not limited to), further described below.
In other embodiments, two Fc domain monomers are included at CH3 domains comprising identical mutations of opposite charge in at least two positions within the loop of charged residues at the interface between the domains. By reversing the charge of both members of two or more complementary pairs of residues in two Fc domain monomers, the mutated Fc domain monomer remains complementary to an Fc domain monomer of the same mutated sequence, but has a lower complementarity to an Fc domain monomer that does not have these mutations. In one embodiment, the Fc domain comprises an Fc monomer comprising the double mutant K409D/D399K, K392D/D399K, E357K/K370E, D356K/K439D, K409E/D399K, K392E/D399K, E357K/K370D, or D356K/K439E. In another embodiment, the Fc domain comprises Fc monomers including four mutants in combination with any double mutant pair, e.g., K409D/D399K/E357K/K370E.
Formation of such Fc domains is by CH3 compatible amino acid substitutions in the constant domains of the antibody. Two dimerization selectivity modules containing incompatible amino acid substitutions (e.g., both containing engineered cavities, both containing engineered protrusions, or both at CH3-CH3 interfaces both contain the same charged amino acid) will not promote Fc domain formation.
In addition, other methods for facilitating the formation of Fc domains with defined Fc domain monomers include, but are not limited to: the LUZ-Y method (U.S. patent application publication No. WO2011034605) which includes the fusion of the C-terminus of the leucine zipper monomer alpha-helices to each Fc domain monomer to allow heterodimer formation, and the chain exchange engineered domain (SEED) host method (Davis) et al, Protein engineering selection (Protein Eng Des Sel.)23:195-202,2010) which generates a polypeptide with C-helices each containing IgA and IgGH3 sequence of an alternating fragment of the sequenceA domain.
Engineered cavities and engineered protrusions
The use of engineered cavities and engineered protrusions (or "protrusion-entry-hole" strategies) is described by Carter (Carter) and colleagues (Ridgway et al, Protein engineering (Protein Eng.)9: 617-. The protuberance and pore interactions contribute to heterodimer formation, while the protuberance-protuberance and pore-pore interactions interfere with homodimer formation due to steric clashes and lack of favorable interactions. This "protrusion-entry-hole" technique is also disclosed in U.S. Pat. No. 5,731,168.
In the present invention, the engineered cavities and engineered protrusions are used to make the Fc constructs described herein. An engineered cavity is a void formed when an original amino acid in a protein is replaced with a different amino acid having a smaller side chain volume. Engineered projections are projections formed when an original amino acid in a protein is replaced with a different amino acid having a larger side chain volume. Specifically, the amino acid being replaced is C in the Fc domain monomerH3 antibody constant domain and is involved in dimerization of two Fc domain monomers. In some embodiments, at one CH3 formation of an engineered cavity in the constant Domain of the antibody to receive another CH3 engineered protrusions in the constant domain of the antibody such that two C' sHThe 3 antibody constant domains both act as dimerization selectivity modules (described above) that promote or contribute to dimerization of the two Fc domain monomers. In other embodiments, at one CH3 formation of an engineered cavity in the antibody constant Domain to better accommodate another CH3 original amino acids in the constant domain of the antibody. In still other embodiments, at one CH3 formation of engineered protrusions in the constant Domain of an antibody to interact with another CH3 the original amino acids in the constant domain of the antibody form additional interactions.
By using amino acids containing smaller side chains (such as C)Amino acids containing larger side chains (such as tyrosine or tryptophan) are replaced with amino acids such as valine, threonine) to construct the engineered cavity. Specifically, some dimerization selectivity modules (described further above) are at CH3 antibody constant domain containing such as Y407V mutation engineered cavity. Similarly, engineered protrusions can be constructed by replacing amino acids containing smaller side chains with amino acids containing larger side chains. Specifically, some dimerization selectivity modules (described further above) are at CH3 antibody constant domains containing engineered protrusions such as the T366W mutation. In the present invention, the engineered cavities and engineered protrusions are also combined with C engineered to enhance heterodimer formationH3 inter-domain disulfide bond combination. Specifically, the cavity Fc contains the Y349C mutation, and the protrusion Fc contains the S354C mutation. Other engineered cavities and engineered protrusions in combination with disulfide-bond engineering or structural calculations (hybrid HA-TF) are included without limitation in table 1.
TABLE 1
Figure BDA0003149346320000211
Replacement of C with a different amino acid residue can be achieved by altering the nucleic acid encoding the original amino acid residueH3 original amino acid residues in the constant domain of the antibody. The upper limit on the number of original amino acid residues that can be replaced, while still maintaining sufficient interaction at the interface, is CH3 total number of residues in the antibody constant domain interface.
V. electrostatic steering
Electrostatic steering is the use of favorable electrostatic interactions between oppositely charged amino acids in peptides, protein domains, and proteins to control the formation of higher order protein molecules. A method of altering the interaction of antibody domains using electrostatic steering effects to reduce homodimer formation in favor of heterodimer formation in bispecific antibody generation is disclosed in U.S. patent application publication No. 2014-0024111.
In the present invention, electrostatic steering is used to control dimerization of the Fc domain monomers and formation of the Fc construct. Specifically, to control dimerization of Fc domain monomers using electrostatic steering, constituent C is replaced with positively or negatively charged amino acid residuesH3-CH3 such that the interaction becomes electrostatically favorable or unfavorable depending on the particular charged amino acid introduced. In some embodiments, an amino acid with a positive charge in the interface (such as lysine, arginine, or histidine) is replaced with a negatively charged amino acid (such as aspartic acid or glutamic acid). In other embodiments, amino acids that are negatively charged in the interface are replaced with amino acids that are positively charged. Charged amino acids can be introduced into interacting CH3 in one or both of the constant domains of the antibody. By introducing charged amino acids into interacting CH3 antibody constant domain, the dimerization selectivity module formed (described further above) can selectively form Fc domain monomer dimers, as controlled by electrostatic steering effects caused by interactions between charged amino acids.
In one embodiment, to form a dimerization selectivity module comprising opposite charges, C is replaced with LysH3 amino acid Asp399 in the constant domain of the antibody and substituting Asp for amino acid Lys 409. Heterodimerization of Fc domain monomers can be promoted by introducing different but compatible mutations in the two Fc domain monomers, such as including but not limited to pairs of charged residues in table 2, and homodimerization of Fc domain monomers can be promoted by introducing the same mutation in the two Fc domain monomers in a symmetric fashion, such as double mutants K409D/D399K or K392D/D399K.
TABLE 2
Figure BDA0003149346320000231
VI. joint
In the present invention, linkers are used to describe polypeptide or protein domains and/orA linkage or linkage between associated non-protein moieties. In some embodiments, the linker is a connection or linkage between at least two Fc domain monomers whose linkers link the C of a first Fc domain monomerH3 the C-terminus of the antibody constant domain is linked to the N-terminus of the hinge domain of the second Fc domain monomer such that the two Fc domain monomers are linked to each other in tandem. In other embodiments, the linker is a connection between the Fc domain monomer and any other protein domain attached thereto. For example, the linker may be C of an Fc domain monomerH3 the C-terminus of the antibody constant domain is attached to the N-terminus of the albumin binding peptide. In another example, the linker may be C H1 the C-terminus of the constant domain of the antibody is linked to the N-terminus of the hinge domain of the Fc domain monomer. In still other embodiments, the linker may connect two single protein domains (excluding the Fc domain), e.g., CLThe C-terminus of the antibody constant domain may be attached to C by a linkerH1N-terminal to the constant domain of the antibody.
The linker may be a simple covalent bond (e.g., a peptide bond), a synthetic polymer (e.g., a polyethylene glycol (PEG) polymer), or any type of bond formed by a chemical reaction (e.g., chemical coupling). In the case where the linker is a peptide bond, a carboxylic acid group at the C-terminus of one protein domain may react with an amino group at the N-terminus of another protein domain in a condensation reaction to form a peptide bond. In particular, peptide bonds can be formed synthetically by conventional organic chemical reactions well known in the art, or by natural production by a host cell, in which a polynucleotide sequence encoding a DNA sequence of two proteins (e.g., two Fc domain monomers) in tandem can be directly transcribed and translated into a contiguous peptide encoding the two proteins by molecular machinery (e.g., DNA polymerases and ribosomes) necessary in the host cell.
Where the linker is a synthetic polymer (e.g., a PEG polymer), the polymer may be functionalized at each end with a reactive chemical functional group to react with the terminal amino acids at the connecting ends of the two proteins.
In the case where the linker (in addition to the peptide bond described above) is formed by a chemical reaction, chemical functional groups (e.g., amine, carboxylic acid, ester, azide, or other functional groups commonly used in the art) may be synthetically attached to the C-terminus of one protein and the N-terminus of another protein, respectively. The two functional groups then react to form a chemical bond by synthetic chemistry, thereby linking the two proteins together. Such chemical coupling procedures are routine to those skilled in the art.
Spacer region
In the present invention, the linker between the two Fc domain monomers may be an amino acid spacer comprising 3-200 amino acids. Suitable peptide spacers are known in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine and serine. In certain embodiments, the spacer may contain a motif, such as multiple motifs or repeated motifs of GS, GGS, GGGGS (SEQ ID NO:1), GGSG (SEQ ID NO:2), or SGGG (SEQ ID NO: 3). In certain embodiments, the spacer may contain 2 to 12 amino acids, including motifs of GS, such as GS, GSGS (SEQ ID NO:4), GSGSGS (SEQ ID NO:5), GSGSGSGS (SEQ ID NO:6), GSGSGSGSGS (SEQ ID NO:7), or GSGSGSGSGSGS (SEQ ID NO: 8). In certain other embodiments, the spacer may contain 3 to 12 amino acids, including motifs of GGS, such as GGS, GGSGGS (SEQ ID NO:9), GGSGGSGGS (SEQ ID NO:10), and GGSGGSGGSGGS (SEQ ID NO: 11). In still other embodiments, the spacer may contain 4 to 12 amino acids, including motifs of GGSG (SEQ ID NO:12), such as GGSG (SEQ ID NO:13), GGSGGGSG (SEQ ID NO:14), or GGSGGGSGGGSG (SEQ ID NO: 15). In other embodiments, the spacer may contain a motif for GGGGS (SEQ ID NO:16), such as GGGGSGGGGSGGS (SEQ ID NO: 17). In other embodiments, the spacer may also contain amino acids other than glycine and serine, such as GENLYFQSGG (SEQ ID NO:18), SACYCELS (SEQ ID NO:19), RSIAT (SEQ ID NO:20), RPACKIPNDLKQKVMNH (SEQ ID NO:21), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO:22), AAANSSIDLISVPVDSR (SEQ ID NO:23), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 24). In certain embodiments of the invention, two Fc domain monomers are linked in tandem using a 12-or 20-amino acid peptide spacer (FIGS. 4-6), which consists of the sequences GGGSGGGSGGGS (SEQ ID NO:25) and SGGGSGGGSGGGSGGGSGGG (SEQ ID NO:26), respectively. In other embodiments, an 18-amino acid peptide spacer consisting of sequence GGSGGGSGGGSGGGSGGS (SEQ ID NO:27) is used to link CLAnd C H1 antibody constant domain (fig. 7A-7B).
Serum protein binding peptides
Binding to a serum protein peptide may improve the pharmacokinetics of the protein drug, and in particular the Fc constructs described herein may have been fused to a serum protein binding peptide.
As one example, albumin binding peptides that can be used in the methods and compositions described herein are generally known in the art. In one embodiment, the albumin binding peptide comprises the sequence DICLPRWGCLW (SEQ ID NO: 28).
In the present invention, the albumin binding peptide may be attached to the N-terminus or C-terminus of certain polypeptides in the Fc construct. In one embodiment, the albumin binding peptide may be attached to the C-terminus of one or more of the polypeptides in constructs 1, 2, 3, or 7A (fig. 1, 2, 3, and 7A, respectively). In another example, the albumin binding peptide may be fused to the C-terminus of a polypeptide encoding two Fc domain monomers linked in tandem in constructs 4, 5, and 6 (fig. 4, 5, and 6, respectively). In yet another example, the albumin binding peptide may be attached to the C-terminus of an Fc domain monomer that is linked to a second Fc domain monomer in a polypeptide encoding two Fc domain monomers linked in tandem, as shown in constructs 4 and 6 (fig. 4 and 6, respectively). The albumin binding peptide may be genetically fused to the Fc construct or attached to the Fc construct by chemical means (e.g., chemical coupling). If desired, a spacer may be inserted between the Fc construct and the albumin binding peptide. Without being bound by theory, it is expected that inclusion of albumin binding peptides in the Fc constructs of the invention may allow for prolonged retention of therapeutic proteins by their binding to serum albumin.
Fc constructs
The present invention relates generally to Fc constructs having 2-10 Fc domains. These Fc constructs can have a binding affinity and/or avidity for an Fc receptor (e.g., Fc γ RIIIa) that is greater than a single wild-type Fc domain. The invention discloses the interaction of two CH3 antibody constant domain interface amino acids engineered so that the two Fc domain monomers of the Fc domain selectively form a dimer with each other, thus preventing the formation of unwanted multimers or aggregates. The Fc construct comprises an even number of Fc domain monomers, wherein each pair of Fc domain monomers forms an Fc domain. The Fc construct comprises at a minimum one functional Fc domain formed from a dimer of two Fc domain monomers.
In some embodiments, the Fc construct contains one Fc domain comprising a dimer of two Fc domain monomers (fig. 1-3 and 7A). Interacting CH3 the antibody constant domains may be unmodified (figure 1) or may contain amino acid substitutions at their interface. Specifically, the amino acid substitution can be an engineered cavity (fig. 2), an engineered protrusion (fig. 2), or a charged amino acid (fig. 3).
In other embodiments, the Fc construct contains two Fc domains formed by three polypeptides (fig. 4 and 6). The first polypeptide contains two Fc domain monomers connected in tandem by a linker, and the second and third polypeptides contain one Fc domain monomer. The second polypeptide and the third polypeptide may be the same polypeptide or may be different polypeptides. Figure 4 depicts an example of such an Fc construct. The first polypeptide contains two wild-type Fc domain monomers connected in tandem by a linker, and the second and third polypeptides each contain one wild-type Fc domain monomer. One Fc domain monomer in the first polypeptide forms a first Fc domain with the second polypeptide and another Fc domain monomer in the first polypeptide forms a second Fc domain with the third polypeptide. The second polypeptide and the third polypeptide are different from each otherAttached or connected. Figure 6 depicts an Fc construct similar to that of figure 4. In FIG. 6, the Fc domain monomers in the first polypeptide are both contained at CH3 an engineered overhang in the constant domain of the antibody, and a second polypeptide and a third polypeptide are contained at CH3 engineered cavities in the antibody constant domain. Engineered protrusion-entry-cavity CH3-CHThe 3 interface contributes to the formation of heterodimers of Fc domain monomers and prevents uncontrolled formation of unwanted multimers. As further described herein, in example 4, engineered C is includedH3 dimerization selectivity module of antibody constant domains prevents the formation of unwanted multimers seen in example 3, which example 3 describes the formation of Fc constructs from Fc domain monomers lacking a dimerization selectivity module.
Furthermore, in other embodiments, the Fc construct may contain three Fc domains formed by four polypeptides (fig. 5). The first and second polypeptides may be the same or different, as may the third and fourth polypeptides. In this example, both the first and second polypeptides encode two Fc domain monomers connected in tandem by a linker, with one Fc domain monomer at CH3 antibody constant Domain contains charged amino acid substitutions and the other Fc Domain monomer is at CH3 the constant domain of the antibody contains a protuberance. Both the third and fourth polypeptides encode an Fc domain monomer having a cavity. The first polypeptide and the second polypeptide are bound by their CH3 the interaction of opposite charges in the constant domains of the antibodies forms a first Fc domain with each other. The second and third Fc domains are formed by protrusion-entry-cavity interaction between protrusions in the first and second polypeptides and cavities in the third and fourth polypeptides. Each Fc domain monomer in this Fc construct contains a dimerization selectivity module that promotes the formation of a particular Fc domain.
In still other embodiments, a single polypeptide can form a dimer (e.g., construct 7A; FIG. 7A) or multimer (e.g., construct 7B; FIG. 7B), not through CH3 between antibody constant domainsInteract with each other but through CLConstant Domain and C H1 interaction between constant domains. FIG. 7B depicts a C containing one of the Fc domainsLC of Domain to Adjacent Fc Domain H1 domain-interacting multiple Fc domain Fc constructs.
In still other embodiments, the Fc construct may contain five Fc domains formed from six polypeptides. Two examples are depicted in fig. 8 and 9. When the depicted Fc constructs comprise six polypeptides, four of the polypeptides may be encoded by the same nucleic acid, and the remaining two polypeptides may also be encoded by the same nucleic acid. As a result, these Fc constructs can be produced by expression of both nucleic acids in a suitable host cell.
In another embodiment, an Fc construct containing two or more Fc domains may be formed from two polypeptides having the same primary sequence. Such a construct may be formed by expression of a single polypeptide sequence in a host cell. One example is depicted in fig. 10. In this example, a single nucleic acid is sufficient to encode an Fc construct containing three Fc domains. Allowing two Fc domain monomers that are part of the same polypeptide to form an Fc domain by including a flexible linker of sufficient length and flexibility; this linker may be a cleavable linker. This same polypeptide also contains a third Fc domain monomer connected by an optional flexible linker. This third Fc domain monomer can be linked to another Fc domain monomer to produce the Y-shaped Fc construct depicted in figure 10. The formation of the Fc domain can be controlled by using a dimerization selectivity module, as also depicted in fig. 10.
IX. host cell and protein production
In the present invention, a host cell refers to a vector that contains the necessary cellular components (e.g., organelles) required for expression of the polypeptides and constructs described herein from their respective nucleic acids. These nucleic acids may be contained in a nucleic acid vector that can be introduced into a host cell by conventional techniques known in the art (transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). The host cell may be of mammalian or bacterial origin. Mammalian host cells include, but are not limited to, CHO (or CHO-derived cell lines such as CHO-K1, CHO-DXB11, CHO-DG44), murine host cells (e.g., NS0, Sp2/0), VERY, HEK (e.g., HEK293), BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, CRL7O3O and HsS78Bst cells. Host cells may also be selected that regulate the expression of the protein construct, or modify and process the protein product in a particular manner as desired. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of protein products. Appropriate cell lines or host systems may be selected to ensure proper modification and processing of the expressed protein.
For expression and secretion of the protein product from their respective DNA plasmid constructs, host cells can be transfected or transformed with DNA under the control of appropriate expression control elements known in the art, including promoters, enhancers, sequences, transcription terminators, polyadenylation sites, and selectable markers. Methods for expressing therapeutic proteins are known in the art. See, e.g., baulina Balbas (Paulina Balbas), aheliia claustrons (Argelia Lorence) (editors) recombinant gene expression: review and protocol (Methods in Molecular Biology), the Homana Press (Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology); second edition 2004 (7 months and 20 days 2004); frakemlvoronov (Vladimir Voynov) and giastin (Justin) a. caravell (caravela) (editorial) therapeutic proteins: methods and Protocols (Therapeutic Proteins: Methods and Protocols) (Methods in molecular biology) Homena Press; the second version 2012 (6 months and 28 days 2012).
X. purification
The Fc construct may be purified by any method known in the art of protein purification, such as by chromatography (e.g., ion exchange, affinity chromatography (e.g., protein a affinity chromatography), and size exclusion column chromatography), centrifugation, differential solubilization, or by any other standard technique for purifying proteins. For example, Fc constructs can be isolated and purified by appropriate selection and combination of affinity columns (such as protein a columns and chromatography columns), filtration, ultrafiltration, salting out, and dialysis procedures (see, e.g., Production Scale Purification of Antibodies (Process Scale Purification of Antibodies), uwei gothsak (editors) John Wiley & Sons, Inc., 2009; and sabo raman (editors) antibody-Volume I-Production and Purification (Antibodies-Volume I-Production and Purification), kruyveromyces/plenanism press publications (Kluwer acaada/plenium papers), new york (2004)). In some cases, the Fc construct may be coupled to a marker sequence, such as a peptide, that facilitates purification. An example of a tag amino acid sequence is a hexa-histidine peptide bound to a nickel-functionalized agarose affinity column with micromolar affinity. Other peptide tags suitable for purification include, but are not limited to, the hemagglutinin "HA" tag corresponding to an epitope derived from the influenza hemagglutinin protein (Wilson et al, 1984, Cell (Cell)37: 767).
For the Fc construct, protein a column chromatography can be used as a purification method. Protein a ligands interact with Fc constructs via the Fc region, making protein a chromatography a highly selective capture method that eliminates most host cell proteins. In the present invention, the Fc construct can be purified using protein a column chromatography as described in example 2.
Xi pharmaceutical composition/preparation
The present invention relates to pharmaceutical compositions comprising one or more Fc constructs described herein. In one embodiment, the pharmaceutical composition comprises a substantially homologous collection of structurally identical or substantially identical Fc constructs. In various examples, the pharmaceutical composition comprises a substantially homologous set of any one of constructs 1-10 and 5.
The therapeutic protein constructs (e.g., Fc constructs) of the invention can be incorporated into pharmaceutical compositions. Pharmaceutical compositions comprising therapeutic proteins may be formulated by methods known to those skilled in the art. The pharmaceutical composition may be administered parenterally in the form of an injectable formulation comprising a sterile solution or suspension in water or another pharmaceutically acceptable liquid. For example, the pharmaceutical composition may be formulated by appropriately combining the Fc construct with a pharmaceutically acceptable carrier or vehicle, such as sterile water for injection (WFI), physiological saline, emulsifiers, suspending agents, surfactants, stabilizers, diluents, binders, excipients, and then mixing in unit dosage forms as required by generally acceptable pharmaceutical practice. The amount of active ingredient contained in the pharmaceutical preparation is such as to provide such a suitable dosage within the specified range.
Sterile compositions for injection may be formulated according to conventional pharmaceutical practice using distilled water for injection as a vehicle. For example, physiological saline or isotonic solutions containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol and sodium chloride may be used as an aqueous injection solution, optionally with a suitable solubilizer, for example alcohols such as ethanol and polyols (such as propylene glycol or polyethylene glycol), and a non-ionic surfactant such as polysorbate 80TMHCO-50, and analog combinations generally known in the art. Methods for formulation of therapeutic protein products are known in the art, see, e.g., Banga (Banga) (editors) therapeutic peptides and proteins: formulating, Processing and Delivery Systems (Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems) (second edition) Taylor-Francisels group CRC Press (Taylor)&Francis Group,CRC Press)(2006)。
XII dosage
The pharmaceutical composition is administered in a manner compatible with the dosage formulation and in such an amount as is therapeutically effective so as to produce an improvement or amelioration of symptoms. The pharmaceutical composition is administered in a variety of dosage forms, for example, intravenous dosage forms, subcutaneous dosage forms, oral dosage forms (such as ingestible solutions, drug release capsules), and the like. The appropriate dosage for an individual subject depends on the purpose of the treatment, the route of administration, and the condition of the patient. In general, the recombinant protein is administered in a dose of 1-200mg/kg, such as 1-100mg/kg, such as 20-100 mg/kg. Therefore, it is essential for the medical provider to adjust and determine the dosage and modify the route of administration as needed to obtain the best therapeutic effect.
Indication xiii
The pharmaceutical compositions and methods of the invention are useful for reducing inflammation in a subject, promoting clearance of autoantibodies in a subject, inhibiting antigen presentation in a subject, reducing an immune response in a subject (e.g., blocking immune response based activation of immune complexes), and treating immunological and inflammatory conditions or diseases in a subject. Exemplary conditions and diseases include Rheumatoid Arthritis (RA); systemic Lupus Erythematosus (SLE); ANCA-associated vasculitis; antiphospholipid antibody syndrome; autoimmune hemolytic anemia; chronic inflammatory demyelinating neuropathies; clearance of anti-allogenic in transplants, anti-autoantibodies in GVHD, anti-surrogate, IgG therapeutic, IgG aberrant proteins; dermatomyositis; goodpasture's syndrome; targeted organ system type II hypersensitivity syndromes mediated by antibody-dependent cell-mediated cytotoxicity, such as gilland barre syndrome, CIDP, dermatomyositis, feldian syndrome, antibody-mediated rejection, autoimmune thyroid disease, ulcerative colitis, autoimmune liver disease; idiopathic thrombocytopenic purpura; myasthenia gravis, neuromyelitis optica; pemphigus and other autoimmune foaming disorders; scleroderma; autoimmune cytopenia and other disorders mediated by antibody-dependent phagocytosis; other FcR-dependent inflammatory syndromes such as synovitis, dermatomyositis, systemic vasculitis, glomerulonephritis and vasculitis.
Detailed Description
The present application also relates to the following embodiments:
1. an Fc construct comprising:
a) a first polypeptide having the formula a-L-B; wherein
i) A comprises a first Fc domain monomer;
ii) L is a linker; and is
B comprises a second Fc domain monomer;
b) a second polypeptide comprising a third Fc domain monomer; and
c) a third polypeptide comprising a fourth Fc domain monomer;
wherein the first and third Fc domain monomers combine to form a first Fc domain and the second and fourth Fc domain monomers combine to form a second Fc domain.
2. The Fc construct of embodiment 1, wherein said first Fc domain monomer and said third Fc domain monomer comprise complementary dimerization selectivity modules that promote dimerization between said first Fc domain monomer and said third Fc domain monomer.
3. The Fc construct of embodiment 1 or 2, wherein said second Fc domain monomer and said fourth Fc domain monomer comprise complementary dimerization selectivity modules that promote dimerization between said second Fc domain monomer and said fourth Fc domain monomer.
4. The Fc construct of any one of embodiments 1-3, wherein a consists of an Fc domain monomer.
5. The Fc construct of any one of embodiments 1-4, wherein B consists of an Fc domain monomer.
6. The Fc construct of any one of embodiments 1-5, wherein said second polypeptide consists of an Fc domain monomer.
7. The Fc construct of any one of embodiments 1-6, wherein said third polypeptide consists of an Fc domain monomer.
8. The Fc construct of any one of embodiments 1-7, further comprising an albumin binding peptide linked to the N-terminus or C-terminus of one or more of the polypeptides by a linker.
9. The Fc construct of any one of embodiments 1-8, further comprising an IgG CLAntibody constant domains and IgG C H1 an antibody constant domain, wherein said IgG C H1 an antibody constant domain is attached to a or the N-terminus of said second polypeptide by a linker.
10. The Fc construct of any one of embodiments 1-9, wherein said second polypeptide and said third polypeptide have the same amino acid sequence.
11. An Fc construct comprising:
a) a first polypeptide having the formula a-L-B; wherein
i) A comprises a first Fc domain monomer;
ii) L is a linker; and is
B comprises a second Fc domain monomer;
b) a second polypeptide having the formula a ' -L ' -B '; wherein
i) A' comprises a third Fc domain monomer;
l' is a linker; and is
B' comprises a fourth Fc domain monomer;
c) a third polypeptide comprising a fifth Fc domain monomer; and
d) a fourth polypeptide comprising a sixth Fc domain monomer;
wherein A of the first polypeptide and A 'of the second polypeptide combine to form a first Fc domain, B of the first polypeptide and a fifth Fc domain monomer combine to form a second Fc domain, and B' of the second polypeptide and a sixth Fc domain monomer combine to form a third Fc domain.
12. The Fc construct of embodiment 11, wherein said first Fc domain monomer and said third Fc domain monomer comprise complementary dimerization selectivity modules that promote dimerization between said first Fc domain monomer and said third Fc domain monomer.
13. The Fc construct of embodiment 11 or 12, wherein said second Fc domain monomer and said fifth Fc domain monomer comprise complementary dimerization selectivity modules that promote dimerization between said second Fc domain monomer and said fifth Fc domain monomer.
14. The Fc construct of any one of embodiments 11-13, wherein said fourth Fc domain monomer and said sixth Fc domain monomer comprise complementary dimerization selectivity modules that promote dimerization between said fourth Fc domain monomer and said sixth Fc domain monomer.
15. The Fc construct of any one of embodiments 11-14, wherein a consists of an Fc domain monomer.
16. The Fc construct of any one of embodiments 11-15, wherein B consists of an Fc domain monomer.
17. The Fc construct of any one of embodiments 11-16, wherein a' consists of an Fc domain monomer.
18. The Fc construct of any one of embodiments 11-17, wherein B' consists of an Fc domain monomer.
19. The Fc construct of any one of embodiments 11-18, wherein said third polypeptide consists of an Fc domain monomer.
20. The Fc construct of any one of embodiments 11-19, wherein said fourth polypeptide consists of an Fc domain monomer.
21. The Fc construct of any one of embodiments 11-20, further comprising IgG CLAntibody constant domains and IgG C H1 an antibody constant domain, wherein said IgG CLAntibody constant domains are attached to the IgG C by linkers H1 the N-terminal end of the constant domain of the antibody, and said IgG C H1 antibody constant domain is attached to the N-terminus of a via a linker.
22. The Fc construct of embodiment 21, further comprising a second IgG CLAntibody constant domains and second IgG C H1 antibody constant domain, wherein the second IgG CLThe antibody constant domain is attached to the second IgG C by a linker H1 the N-terminus of the constant domain of the antibody, and said second IgG C H1 an antibody constant domain is attached to the N-terminus of A' via a linker,
the IgG C H1 antibody constant domains are attached to the N-terminus of a or a' via a linker.
23. The Fc construct of any one of embodiments 11-21, further comprising an albumin binding peptide.
24. The Fc construct of any one of embodiments 11-22, wherein said first polypeptide and said second polypeptide have the same amino acid sequence and wherein said third polypeptide and said fourth polypeptide have the same amino acid sequence.
25. An Fc construct comprising:
a) a first polypeptide having the formula a-L-B; wherein
i) A comprises a first Fc domain monomer;
ii) L is a linker; and is
B) comprises an albumin binding peptide; and
b) a second polypeptide comprising a second Fc domain monomer;
wherein the first Fc domain monomer and the second Fc domain monomer combine to form an Fc domain.
26. The Fc construct of embodiment 25, wherein said first Fc domain monomer and said second Fc domain monomer comprise complementary dimerization selectivity modules that promote dimerization between said first Fc domain monomer and said second Fc domain monomer.
27. The Fc construct of embodiment 25 or 26, wherein a consists of an Fc domain monomer.
28. The Fc construct of any one of embodiments 25-27, wherein the second polypeptide consists of an Fc domain monomer.
29. An Fc construct comprising:
a) a first polypeptide having the formula a-L1-B-L2-C; wherein
i) A comprises IgG CLAn antibody constant domain;
ii), L1 and L2 are each linkers;
iii) B comprises IgG C H1 an antibody constant domain; and is
C comprises a first Fc domain monomer; and
b) a second polypeptide having the formula a ' -L1 ' -B ' -L2 ' -C '; wherein
i) A' comprises IgG CLAn antibody constant domain;
ii), L1 'and L2' are each linkers;
iii) B' comprises IgG C H1 an antibody constant domain; and is
C) comprises a second Fc domain monomer; wherein
The first Fc domain monomer and the second Fc domain monomer combine to form an Fc domain.
30. The Fc construct of embodiment 29, wherein said first Fc domain monomer and said second Fc domain monomer comprise complementary dimerization selectivity modules that promote dimerization between said first Fc domain monomer and said second Fc domain monomer.
31. The Fc construct of embodiment 29 or 30, wherein C consists of an Fc domain monomer.
32. The Fc construct of any one of embodiments 29-31, wherein C' consists of an Fc domain monomer.
33. The Fc construct of any one of embodiments 29-32, further comprising an albumin binding peptide linked to the C-terminus of C or C' by a linker.
34. An Fc construct comprising:
a) a first polypeptide having the formula a-L1-B-L2-C; wherein
i) A comprises IgG CLAn antibody constant domain;
ii), L1 and L2 are each linkers;
iii) B comprises IgG C H1 an antibody constant domain; and is
C comprises a first Fc domain monomer; and
b) a second polypeptide having the formula a ' -L1 ' -B ' -L2 ' -C '; wherein
i) A' comprises IgG CLAn antibody constant domain;
ii), L1 'and L2' are each linkers;
iii) B' comprises IgG C H1 an antibody constant domain; and is
C) comprises a second Fc domain monomer;
c) a third polypeptide comprising a third Fc domain monomer; and
d) a fourth polypeptide comprising a fourth Fc domain monomer; wherein
The first Fc domain monomer and the third Fc domain monomer combine to form a first Fc domain;
the second Fc domain monomer and a fourth third Fc domain monomer combine to form a second Fc domain;
the IgG C of the first polypeptide H1 constant Domain of an antibody and the IgG C of said second polypeptideLAntibody constant domain combinations; and is
The IgG C of said second polypeptide H1 antibody constant domain and said IgG C of said first polypeptideLAntibody constant domain combinations.
35. An Fc construct comprising:
a) a first polypeptide comprising a first Fc domain monomer; and
b) a second polypeptide comprising a second Fc domain monomer; wherein
The first and second Fc domain monomers combine to form an Fc domain, and the first and second Fc domain monomers comprise complementary dimerization selectivity modules that promote dimerization between the first and second Fc domain monomers.
36. The Fc construct of embodiment 35, wherein said first polypeptide consists of an Fc domain monomer.
37. The Fc construct of embodiment 35 or 36, wherein said second polypeptide consists of an Fc domain monomer.
38. The Fc construct of any one of embodiments 35-37, further comprising an albumin binding peptide.
39. The Fc construct of any one of embodiments 35-38, further comprising IgG CLAntibody constant domains and IgG C H1 an antibody constant domain, wherein said IgG C H1 an antibody constant domain is attached to the N-terminus of the first polypeptide or the second polypeptide by a linker.
40. An Fc construct comprising:
a) a first polypeptide having the formula a-L-B; wherein
i) A comprises a first Fc domain monomer;
ii) L is a linker; and is
B comprises a second Fc domain monomer;
b) a second polypeptide having the formula a ' -L ' -B '; wherein
i) A' comprises a third Fc domain monomer;
l' is a linker; and is
B' comprises a fourth Fc domain monomer;
wherein the first and second Fc domain monomers are comprised at their corresponding CH3 domain, and the third and fourth Fc domain monomers comprise C in their respectiveH3, wherein the engineered cavity and the engineered protuberance are positioned to form a protuberance-entry-cavity pair, wherein the first Fc domain monomer and the third Fc domain monomer combine to form a first Fc domain, and the second Fc domain monomer and the fourth Fc domain monomer combine to form a second Fc domain.
41. The Fc construct of embodiment 40, wherein said A, B, A ', or B' each consists of an Fc domain monomer.
42. The Fc construct of embodiment 40 or 41, further comprising an albumin binding peptide linked to the C-terminus of B or B' by a linker.
43. The Fc construct of any one of embodiments 40-42, further comprising IgG CLAntibody constant domains and IgG C H1 an antibody constant domain, wherein said IgG C H1 antibody constant domains are attached to the N-terminus of a or a' via a linker.
44. The Fc construct of any one of embodiments 2, 3, 12, 13, 14, 26, 30, or 35, wherein said dimerization selectivity module comprises the C in one of said Fc domain monomersH3 domain and the C in another of the Fc domain monomersH3 engineered protrusions in a structural domain, wherein the engineered cavitiesAnd the engineered protrusions are positioned to form a protrusion-entry-cavity pair of Fc domain monomers.
45. The Fc construct of embodiment 44, wherein one said Fc domain monomer comprises Y407V and Y349C and the other said Fc domain monomer comprises T366W and S354C.
46. The Fc construct of any one of embodiments 2, 3, 12, 13, 14, 26, 30, or 35, wherein said dimerization selectivity module comprises the C in one of said Fc domain monomersH3 domain and the C at the other of said Fc domain monomersH3 domain, wherein the negatively charged amino acid and the positively charged amino acid are positioned to facilitate formation of the Fc domain.
47. The Fc construct of embodiment 46, wherein one said Fc domain monomer comprises D399K and the other said Fc domain monomer comprises K409D.
48. The Fc construct of any one of embodiments 1-47, wherein one or more linkers in said Fc construct is a bond.
49. The Fc construct of any one of embodiments 1-47, wherein one or more linkers in said Fc construct is a spacer.
50. The Fc construct of embodiment 49, wherein said spacer comprises the sequence of any one of SEQ ID NOs 1-27.
51. The Fc construct of embodiment 50, wherein said sequence consists of any one of SEQ ID NOs 1, 2, and 3.
52. The Fc construct of any one of embodiments 8, 22, 25, 33, 38, and 42, wherein said albumin binding peptide comprises the sequence of SEQ ID NO: 28.
53. The Fc construct of embodiment 52, wherein said albumin binding peptide consists of the sequence of SEQ ID NO 28.
54. An Fc construct comprising:
a) a first polypeptide having the formula a-L1-B-L2-C; wherein
i) A comprises a first Fc domain monomer;
ii) L1 is a linker;
b comprises a second Fc domain monomer;
iv) L2 is a linker;
v) C comprises a third Fc domain monomer; and
b) a second polypeptide having the formula a ' -L1 ' -B ' -L2 ' -C '; wherein
i) A' comprises a fourth Fc domain monomer;
ii) L1' is a linker;
iii) B' comprises a fifth Fc domain monomer;
iv) L2' is a linker;
v) C' comprises a sixth Fc domain monomer; wherein
The first Fc domain monomer and the second Fc domain monomer combine to form a first Fc domain,
the fourth Fc domain monomer and the fifth Fc domain monomer combine to form a second Fc domain, and the third Fc domain monomer and the sixth Fc domain monomer combine to form a third Fc domain.
55. The Fc construct of embodiment 54, wherein linkers L1 and L1' are cleavable linkers.
56. The Fc construct of any one of embodiments 1-55, wherein one or more of said Fc domain monomers comprises an IgG hinge domain, an IgG C H2 antibody constant Domain, and IgG CH3 an antibody constant domain.
57. The Fc construct of embodiment 56, wherein said Fc domain monomers each comprise an IgG hinge domain, an IgG C H2 antibody constant Domain, and IgG CH3 an antibody constant domain.
58. The Fc construct of any one of embodiments 56 or 57, wherein said IgG has a subtype selected from the group consisting of IgG1, IgG2a, IgG2b, IgG3, and IgG 4.
59. A method of making an Fc construct according to any one of embodiments 1-58, the method comprising:
a) providing a host cell comprising a polynucleotide encoding the polypeptide;
b) expressing said first, second and third polypeptides in said host cell under conditions that allow formation of said Fc construct; and is
c) Recovering the Fc construct.
60. A host cell expressing the Fc construct of any one of embodiments 1-58, said host cell comprising a polynucleotide encoding said polypeptide, wherein said polynucleotide is expressed in said host cell.
61. A pharmaceutical composition comprising a substantially homologous collection of Fc constructs having 2-10 Fc domains.
62. The pharmaceutical composition of embodiment 61, wherein a plurality of the 2-10 Fc domains are formed by association of two Fc domain monomers comprising complementary dimerization selectivity modules that promote dimerization between the two Fc domain monomers.
63. The pharmaceutical composition of embodiment 62, wherein two individual polypeptides in the plurality have the same amino acid sequence.
64. The pharmaceutical composition of embodiment 62, wherein two individual polypeptides in the plurality have different amino acid sequences.
65. The pharmaceutical composition of embodiment 62, wherein the complementary Fc monomer sequences have complementary dimerization selectivity modules.
66. The pharmaceutical composition of embodiment 61, wherein the Fc construct has one or more (e.g., 2, 3, 4, 5, or 6) of the following characteristics:
a) formed at least in part by the association of Fc domain monomers present in different polypeptides;
b) does not comprise additional domains that facilitate the association of the two polypeptides;
c) comprises a covalent bond only between two Fc domain monomers linked to form an Fc domain;
d) no covalent bonds are included between the Fc domains;
e) providing sufficient structural flexibility such that all or substantially all of the Fc domains in the Fc construct are capable of simultaneously interacting with Fc receptors on the cell surface; and/or
f) Comprising at least two Fc domains connected by a linker.
67. A pharmaceutical composition comprising a substantially homologous collection of Fc constructs of any one of embodiments 1-58.
68. The pharmaceutical composition of any one of embodiments 61-67, wherein the Fc construct comprises 2-5 associated polypeptides, each polypeptide comprising at least one Fc domain monomer, wherein the respective Fc domain monomers of the construct are the same or differ by no more than 10 amino acids.
69. The pharmaceutical composition of any one of embodiments 61-67, wherein the Fc construct comprises two Fc domains comprising three associated polypeptides, one of the three polypeptides comprises two Fc domain monomers and two of the three polypeptides each comprise one Fc domain monomer.
70. The pharmaceutical composition of any one of embodiments 61-67, wherein the Fc construct comprises three Fc domains comprising four associated polypeptides, two of the four polypeptides each comprising two Fc domain monomers and two of the four polypeptides each comprising one Fc domain monomer.
71. The pharmaceutical composition of any one of embodiments 61-67, wherein the Fc construct consists of n Fc domains comprising 2n polypeptides, each polypeptide comprising an Fc domain monomer, IgG CLAntibody constant domain, and IgG C H1 antibody constant domain.
72. A method of treating inflammation in a subject, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an Fc construct of any one of embodiments 1-58 or a pharmaceutical composition of any one of embodiments 61-71.
73. A method of reducing immune response immune complex-based activation in a subject, the method comprising administering to the subject an Fc construct according to any one of embodiments 1-58 or a pharmaceutical composition according to any one of embodiments 61-71.
74. The method of embodiment 73, wherein the subject has an autoimmune disease.
75. An Fc construct consisting of
a) A first polypeptide having the formula a-L-B; wherein
i) A comprises a first Fc domain monomer;
ii) L is a linker; and is
B comprises a second Fc domain monomer;
b) a second polypeptide having the formula a ' -L ' -B '; wherein
i) A' comprises a third Fc domain monomer;
l' is a linker; and is
B' comprises a fourth Fc domain monomer;
c) a third polypeptide comprising a fifth Fc domain monomer; and
d) a fourth polypeptide comprising a sixth Fc domain monomer;
wherein a and a 'combine to form a first Fc domain, B and the fifth Fc domain monomer combine to form a second Fc domain, and B' and the sixth Fc domain monomer combine to form a third Fc domain;
wherein the first Fc domain monomer and the third Fc domain monomer each comprise a complementary dimerization selectivity module that promotes dimerization between the first Fc domain monomer and the third Fc domain monomer,
the second and fifth Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the second and fifth Fc domain monomers, and
the fourth and sixth Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the fourth and sixth Fc domain monomers, and
wherein the Fc construct contains no more than three Fc domains.
76. An Fc construct consisting of
a) A first polypeptide having the formula a-L-B; wherein
i) A consists of a first Fc domain monomer;
ii) L is a linker; and is
B consists of a second Fc domain monomer;
b) a second polypeptide having the formula a ' -L ' -B '; wherein
i) A' consists of a third Fc domain monomer;
l' is a linker; and is
B' consists of a fourth Fc domain monomer;
c) a third polypeptide consisting of a fifth Fc domain monomer; and
d) a fourth polypeptide consisting of a sixth Fc domain monomer;
wherein a and a 'combine to form a first Fc domain, B and the fifth Fc domain monomer combine to form a second Fc domain, and B' and the sixth Fc domain monomer combine to form a third Fc domain;
wherein the first Fc domain monomer and the third Fc domain monomer each comprise a complementary dimerization selectivity module that promotes dimerization between the first Fc domain monomer and the third Fc domain monomer,
the second and fifth Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the second and fifth Fc domain monomers, and
the fourth and sixth Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the fourth and sixth Fc domain monomers, and
wherein the Fc construct contains no more than three Fc domains.
77. The Fc construct of embodiment 75 or 76, wherein said complementary dimerization selectivity module of said first Fc domain monomer comprises a negatively charged amino acid substitution, said complementary dimerization selectivity module of said third Fc domain monomer comprises a positively charged amino acid substitution, said complementary dimerization selectivity module of said second Fc domain monomer comprises an engineered protrusion, said complementary dimerization selectivity module of said fifth Fc domain monomer comprises an engineered cavity, said complementary dimerization selectivity module of said fourth Fc domain monomer comprises an engineered protrusion, and said complementary dimerization selectivity module of said sixth Fc domain monomer comprises an engineered cavity.
78. The Fc construct of any one of embodiments 75-77, wherein said linker L and/or L' is 3-200 amino acids in length.
79. The Fc construct of embodiment 77, wherein said linker L and/or L' consists of the sequence of any one of SEQ ID NOs 1, 2 and 3.
80. An Fc construct consisting of
a) A first polypeptide having the formula a-L1-B-L2-C; wherein
i) A comprises a first Fc domain monomer;
ii) L1 is a linker;
b comprises a second Fc domain monomer; and is
iv) L2 is a linker;
v) C comprises a third Fc domain monomer; and
b) a second polypeptide having the formula a ' -L1 ' -B ' -L2 ' -C '; wherein
i) A' comprises a fourth Fc domain monomer;
ii) L1' is a linker;
iii) B' comprises a fifth Fc domain monomer; and is
iv) L2' is a linker;
v) C' comprises a sixth Fc domain monomer;
c) a third polypeptide comprising a seventh Fc domain monomer;
d) a fourth polypeptide comprising an eighth Fc domain monomer;
e) a fifth polypeptide comprising a ninth Fc domain monomer;
d) a sixth polypeptide comprising a tenth Fc domain monomer;
wherein a and the seventh Fc domain monomer combine to form a first Fc domain, B and B ' combine to form a second Fc domain, C and the eighth Fc domain monomer combine to form a third Fc domain, a ' and the ninth Fc domain monomer combine to form a fourth Fc domain, and C ' and the tenth Fc domain monomer combine to form a fifth Fc domain;
wherein the first Fc domain monomer and the seventh Fc domain monomer each comprise a complementary dimerization selectivity module that promotes dimerization between the first Fc domain monomer and the seventh Fc domain monomer,
the second and fifth Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the second Fc domain monomer and the fifth Fc domain monomer,
the third and eighth Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the third Fc domain monomer and the eighth Fc domain monomer;
the fourth and ninth Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the fourth and ninth Fc domain monomers, and
the sixth and tenth Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the sixth and tenth Fc domain monomers, and
wherein the Fc construct contains no more than five Fc domains.
81. An Fc construct consisting of
a) A first polypeptide having the formula a-L1-B-L2-C; wherein
i) A consists of a first Fc domain monomer;
ii) L1 is a linker;
b consists of a second Fc domain monomer; and is
iv) L2 is a linker;
v) C consists of a third Fc domain monomer; and
b) a second polypeptide having the formula a ' -L1 ' -B ' -L2 ' -C '; wherein
i) A' consists of a fourth Fc domain monomer;
ii) L1' is a linker;
b' consists of a fifth Fc domain monomer; and is
iv) L2' is a linker;
v) C' consists of a sixth Fc domain monomer;
c) a third polypeptide consisting of a seventh Fc domain monomer;
d) a fourth polypeptide consisting of an eighth Fc domain monomer;
e) a fifth polypeptide consisting of a ninth Fc domain monomer;
d) a sixth polypeptide consisting of a tenth Fc domain monomer;
wherein a and the seventh Fc domain monomer combine to form a first Fc domain, B and B ' combine to form a second Fc domain, C and the eighth Fc domain monomer combine to form a third Fc domain, a ' and the ninth Fc domain monomer combine to form a fourth Fc domain, and C ' and the tenth Fc domain monomer combine to form a fifth Fc domain;
wherein the first Fc domain monomer and the seventh Fc domain monomer each comprise a complementary dimerization selectivity module that promotes dimerization between the first Fc domain monomer and the seventh Fc domain monomer,
the second and fifth Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the second Fc domain monomer and the fifth Fc domain monomer,
the third and eighth Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the third Fc domain monomer and the eighth Fc domain monomer;
the fourth and ninth Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the fourth and ninth Fc domain monomers, and
the sixth and tenth Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the sixth and tenth Fc domain monomers, and
wherein the Fc construct contains no more than five Fc domains.
82. The Fc construct of embodiment 80 or 81, wherein said complementary dimerization selectivity module of the second Fc domain monomer comprises a negatively charged amino acid substitution, said complementary dimerization selectivity module of the fifth Fc domain monomer comprises a positively charged amino acid substitution, said complementary dimerization selectivity modules of each of the first, third, fourth, and sixth Fc domain monomers comprise an engineered protrusion, and said complementary dimerization selectivity modules of each of the seventh, eighth, ninth, and tenth Fc domain monomers comprise an engineered cavity.
83. An Fc construct consisting of
a) A first polypeptide having the formula a-L1-B-L2-C; wherein
i) A comprises a first Fc domain monomer;
ii) L1 is a linker;
b comprises a second Fc domain monomer; and is
iv) L2 is a linker;
v) C comprises a third Fc domain monomer; and
b) a second polypeptide having the formula a ' -L1 ' -B ' -L2 ' -C '; wherein
i) A' comprises a fourth Fc domain monomer;
ii) L1' is a linker;
iii) B' comprises a fifth Fc domain monomer; and is
iv) L2' is a linker;
v) C' comprises a sixth Fc domain monomer;
c) a third polypeptide comprising a seventh Fc domain monomer;
d) a fourth polypeptide comprising an eighth Fc domain monomer;
e) a fifth polypeptide comprising a ninth Fc domain monomer;
d) a sixth polypeptide comprising a tenth Fc domain monomer;
wherein a and a ' combine to form a first Fc domain, B and the seventh Fc domain monomer combine to form a second Fc domain, C and the eighth Fc domain monomer combine to form a third Fc domain, B ' and the ninth Fc domain monomer combine to form a fourth Fc domain, and C ' and the tenth Fc domain monomer combine to form a fifth Fc domain;
wherein the first Fc domain monomer and the fourth Fc domain monomer each comprise a complementary dimerization selectivity module that promotes dimerization between the first Fc domain monomer and the fourth Fc domain monomer,
the second and seventh Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the second Fc domain monomer and the seventh Fc domain monomer,
the third and eighth Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the third Fc domain monomer and the eighth Fc domain monomer;
the fifth and ninth Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the fifth and ninth Fc domain monomers, and
the sixth and tenth Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the sixth and tenth Fc domain monomers, and
wherein the Fc construct contains no more than five Fc domains.
84. An Fc construct consisting of
a) A first polypeptide having the formula a-L1-B-L2-C; wherein
i) A consists of a first Fc domain monomer;
ii) L1 is a linker;
b consists of a second Fc domain monomer; and is
iv) L2 is a linker;
v) C consists of a third Fc domain monomer; and
b) a second polypeptide having the formula a ' -L1 ' -B ' -L2 ' -C '; wherein
i) A' consists of a fourth Fc domain monomer;
ii) L1' is a linker;
b' consists of a fifth Fc domain monomer; and is
iv) L2' is a linker;
v) C' consists of a sixth Fc domain monomer;
c) a third polypeptide consisting of a seventh Fc domain monomer;
d) a fourth polypeptide consisting of an eighth Fc domain monomer;
e) a fifth polypeptide consisting of a ninth Fc domain monomer;
d) a sixth polypeptide consisting of a tenth Fc domain monomer;
wherein a and a ' combine to form a first Fc domain, B and the seventh Fc domain monomer combine to form a second Fc domain, C and the eighth Fc domain monomer combine to form a third Fc domain, B ' and the ninth Fc domain monomer combine to form a fourth Fc domain, and C ' and the tenth Fc domain monomer combine to form a fifth Fc domain;
wherein the first Fc domain monomer and the fourth Fc domain monomer each comprise a complementary dimerization selectivity module that promotes dimerization between the first Fc domain monomer and the fourth Fc domain monomer,
the second and seventh Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the second Fc domain monomer and the seventh Fc domain monomer,
the third and eighth Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the third Fc domain monomer and the eighth Fc domain monomer;
the fifth and ninth Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the fifth and ninth Fc domain monomers, and
the sixth and tenth Fc domain monomers each comprise a complementary dimerization selectivity module that promotes dimerization between the sixth and tenth Fc domain monomers, and
wherein the Fc construct contains no more than five Fc domains.
85. The Fc construct of embodiment 83 or 84, wherein said complementary dimerization selectivity module of the first Fc domain monomer comprises a negatively charged amino acid substitution, said complementary dimerization selectivity module of the fourth Fc domain monomer comprises a positively charged amino acid substitution, said complementary dimerization selectivity modules of each of the second, third, fifth, and sixth Fc domain monomers comprise an engineered protrusion, and said complementary dimerization selectivity modules of each of the seventh, eighth, ninth, and tenth Fc domain monomers comprise an engineered cavity.
86. The Fc construct of any one of embodiments 80-85, wherein said linker L1, L2, L1 ', and/or L2' is 3-200 amino acids in length.
87. The Fc construct of embodiment 86, wherein said linker L1, L2, L1 'and/or L2' consists of the sequence of any one of SEQ ID NOs 1, 2 and 3.
88. An Fc construct of any one of embodiments 1-58 or 75-87 for use in promoting clearance of autoantibodies, inhibiting antigen presentation, reducing an immune response, or reducing immune complex-based activation of the immune response in a subject in need thereof.
89. An Fc construct as described in any one of embodiments 1-58 or 75-87 for use in treating an inflammatory or autoimmune disease or immune disease (e.g., Rheumatoid Arthritis (RA); Systemic Lupus Erythematosus (SLE); ANCA-associated vasculitis; antiphospholipid antibody syndrome; autoimmune hemolytic anemia; chronic inflammatory demyelinating neuropathy; anti-allogenic in transplants, anti-autoantibodies in GVHD, anti-surrogate, IgG therapeutic agents, clearance of IgG abnormal proteins; dermatomyositis; Goodpasture's syndrome; targeted organ system type II hypersensitivity syndrome mediated by antibody-dependent cell-mediated cytotoxicity such as Gillen-Barre syndrome, CIDP, dermatomyositis, Filler's syndrome, antibody-mediated rejection, autoimmune thyroid disease, ulcerative colitis, Autoimmune liver disease; idiopathic thrombocytopenic purpura; myasthenia gravis, neuromyelitis optica; pemphigus and other autoimmune foaming disorders; scleroderma; autoimmune cytopenia and other disorders mediated by antibody-dependent phagocytosis; other FcR-dependent inflammatory syndromes, e.g., synovitis, dermatomyositis, systemic vasculitis, glomerulonephritis, and vasculitis).
Examples of the invention
EXAMPLE 1 design and cloning of DNA plasmid constructs
A total of eight DNA plasmid constructs were used to assemble eight Fc constructs (fig. 1-7B). The DNA plasmid constructs were transfected into Human Embryonic Kidney (HEK)293 cells for protein production. The eight encoded secreted polypeptides have the general structure as described below:
wt Fc: wild-type Fc domain monomers (FIGS. 1: 102 and 104; FIGS. 4: 408 and 410).
B. Overhang Fc: at CH3 Fc domain monomer with engineered overhang in the antibody constant domain (FIG. 2: 202).
C. Cavity Fc: at CH3 Fc domain monomers with engineered cavities in the antibody constant domain (FIG. 2: 204; FIG. 5: 514 and 516).
Cavity Fc: at CH3 Fc domain monomers with engineered cavities in the antibody constant domain (FIG. 2: 204; FIG. 5: 514 and 516). Cavity Fc further contains additional amino acid substitutions relative to cavity Fc.
D. Charge Fc: at CH3 Fc domain monomers with opposite charges in the constant domain of the antibody (FIGS. 3: 302 and 304).
Wt-12-wt Fc 2: two Fc domain monomers connected in tandem by a 12-amino acid GGGS peptide linker (FIG. 4: 402).
F. Overhang-20-charge Fc 2: linked in tandem via a 20-amino acid SGGG peptide linker at CH3 Fc domain monomers with opposite charges in the constant domain of the antibody andH3 Fc domain monomers with engineered overhangs in the antibody constant domain (FIGS. 5: 502 and 508).
F, overhang-20-charge Fc 2: fc domain monomers with opposite charges in the constant domain of the CH3 antibody and Fc domain monomers at C linked in tandem by a 20-amino acid SGGG peptide linkerH3 Fc domain monomers with engineered overhangs in the antibody constant domain (FIGS. 5: 502 and 508). Overhang-20-charge Fc2 contains additional amino acid substitutions relative to overhang Fc.
G. Overhang-20-overhang Fc 2: linked in tandem at C by a 20-amino acid GGGS peptide linkerH3 antibody constant domains both have two Fc domain monomers of engineered protrusions (FIG. 6: 602).
H.CHCLFc +: attached to hinge domain having C H1 and CLConstantFc domain monomers of the domains (FIGS. 7A: 702 and 704; FIGS. 7B: 706, 708, 710, 712, 714, and 716). CLThe constant domain is attached to C by an 18 amino acid GGGS peptide linkerH1A constant domain.
The Fc DNA sequence was derived from human IgG1 Fc. Substitutions for overhang, cavity and charge mutations in the parent Fc sequence. DNA encoding a leader peptide derived from human immunoglobulin kappa light chain was attached to the 5' region. All polypeptides except one (C)HCLFc +) contains this encoded peptide on the amino terminus to direct translocation of the protein to the endoplasmic reticulum for assembly and secretion. It is understood that any of a variety of leader peptides may be used in conjunction with the present invention. The leader peptide is typically cleaved off in the ER lumen. An 11 nucleotide sequence containing a 5' terminal EcoR1 site was added upstream of the ATG start codon. A 30 nucleotide sequence containing a3 'terminal Xho1 site was added downstream of the 3' terminal TGA translation stop codon. These DNA sequences were optimized for expression in mammalian cells and cloned into pcdna3.4 mammalian expression vectors.
Mutations are represented by wild-type amino acid residues followed by positions using the EU kabat numbering system (kabat et al, protein sequences of immunological interest, national institute of health, betesday, maryland, 5 th edition, 1991) and then by a single letter code for the replacement residue. The nucleotide and amino acid sequences of the secreted polypeptides a-H described above are provided below (except for the cavity Fc and overhang-20-charge Fc2 for which only the amino acid sequence is provided).
wt Fc
SEQ ID NO:29:
Figure BDA0003149346320000511
SEQ ID NO:30:
Figure BDA0003149346320000512
Overhang Fc
SEQ ID NO:31:
Figure BDA0003149346320000513
SEQ ID NO:32:
Figure BDA0003149346320000521
Cavity Fc
SEQ ID NO:33:
Figure BDA0003149346320000522
SEQ ID NO:34:
Figure BDA0003149346320000523
Cavity Fc SEQ ID NO 45:
Figure BDA0003149346320000531
charge Fc
SEQ ID NO:35:
Figure BDA0003149346320000532
SEQ ID NO:36:
Figure BDA0003149346320000533
wt-12-wt Fc2
SEQ ID NO:37:
Figure BDA0003149346320000541
SEQ ID NO:38:
Figure BDA0003149346320000542
overhang-20-Charge Fc2
SEQ ID NO:39:
Figure BDA0003149346320000551
SEQ ID NO:40:
Figure BDA0003149346320000552
overhang-20-Charge Fc2
SEQ ID NO:46:
Figure BDA0003149346320000561
Overhang-20-overhang Fc2
SEQ ID NO:41：
Figure BDA0003149346320000562
SEQ ID NO:42：
Figure BDA0003149346320000571
CHCL Fc+
SEQ ID NO:43：
Figure BDA0003149346320000572
SEQ ID NO:44：
Figure BDA0003149346320000581
Example 2 expression of Fc construct proteins
For protein expression of the Fc constructs, two DNA plasmid constructs selected from a-H described in example 1 were transfected into EXPI293 cells (life technologies). Plasmid DNA was introduced into EXPI293 cells using lipofection. The total amount of transfected plasmid constructs was fixed, while the ratio of different plasmid constructs was changed to maximize the yield of the desired constructs (see table 3 below). The ratio (by mass) of the two transfected DNA plasmid constructs for each Fc construct is shown in table 3. Schematic representations of these constructs are shown in fig. 1-7B.
After protein expression, the expressed construct was purified from the cell culture supernatant by protein a-based affinity column chromatography. The culture supernatant was loaded onto a Poros mabscapure a (Life technologies) column using an AKTA Avant preparative chromatography system (GE Healthcare Life Sciences). The captured Fc construct was then washed with phosphate buffered saline (low salt wash) followed by phosphate buffered saline supplemented with 500mM NaCl (high salt wash). The Fc construct was eluted with 100mM glycine, 150mM NaCl, pH 3 buffer. The protein solution coming out of the column was neutralized by adding 1M TRIS, pH 7.4, to a final concentration of 100 mM. By using
Figure BDA0003149346320000582
The XS resin was subjected to ion exchange chromatography to further fractionate the Fc construct (bioscience catalog #4404336 applied). The column was pre-equilibrated with 10mM MES at pH 6 (buffer a) and the sample was eluted with a gradient of 10mM MES, 500mM sodium chloride (buffer B) to pH 6.
A total of seven Fc constructs were obtained (see table 3 below and figures 1-7B). The purified Fc construct was analyzed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) under both reducing and non-reducing conditions, followed by coomassie brilliant blue staining to confirm the presence of a protein band of the expected size.
TABLE 3
Figure BDA0003149346320000591
Example 3-preparation of construct 4 and SDS-PAGE analysis
Construct 4 (FIG. 4) was expressed using two DNA plasmid constructs wt-12-wt Fc2 (DNA plasmid construct E in example 1) and wt Fc (DNA plasmid construct A in example 1). Two plasmid constructs were transfected into HEK293 cells for protein expression and purification as described in example 2. FIGS. 11A-11B show reducing and non-reducing SDS-PAGE of construct 4. On reducing SDS-PAGE (FIG. 11A), a band of approximately 25kDa corresponding to the wt Fc domain monomer (lanes 2 and 3, FIG. 11A) and a band of 50kDa corresponding to the wt-12-wt Fc2 tandem dimer (lanes 1-3, FIG. 11A) were observed. Lanes 2 and 3 each contained the final protein product of construct 4 at higher (1/2) and lower (1/3) protein masses, respectively, on non-reducing SDS-PAGE (fig. 11B). One major band of about 100kDa corresponding to the association of the wt-12-wt Fc2 tandem dimer and two wt Fc domain monomers to form construct 4 was observed, and another major band of about 50kDa with approximately equal signal intensity corresponding to the free wt-12-wt Fc2 tandem dimer not linked to the wt Fc domain monomer.
In addition, higher molecular weight bands of approximately 150kDa, 200kDa and 250kDa were observed corresponding to multimers of wt-12-wt Fc2 and wt Fc domain monomers (lanes 2 and 3, FIG. 11B).
Example 4-preparation of construct 6 and SDS-PAGE analysis
Construct 6 (figure 6) was expressed using two plasmid constructs, projection-20-projection Fc2 (DNA plasmid construct G in example 1) and cavity Fc (DNA plasmid construct C in example 1). Two plasmid constructs were transfected into HEK293 cells for protein expression and purification as described in example 2. FIGS. 12A-12B show reducing and non-reducing SDS-PAGE for construct 6. On reducing SDS-PAGE (FIG. 12A), a band of approximately 25kDa corresponding to the cavity Fc domain monomer (lanes 2 and 3, FIG. 12A) and a band of 50kDa corresponding to the pro-20-pro-Fc 2 (lanes 1-3, FIG. 12A) were observed. Lanes 2 and 3 each contained the final protein product of construct 6 at higher (1/2) and lower (1/3) protein masses on non-reducing SDS-PAGE (FIG. 12B). A major band of about 100kDa corresponding to the association of the knob-20-knob Fc2 tandem dimer and two cavity Fc domain monomers was observed, and a minor band of weaker signal strength at about 50kDa corresponding to the free knob-20-knob Fc2 tandem dimer not combined with any cavity Fc domain monomers.
A similar experiment was performed with construct 5 (fig. 13). Construct 5 (fig. 5) was expressed using two plasmid constructs, overhang-20-charge Fc2 (DNA plasmid construct F in example 1) and cavity Fc (DNA plasmid construct C in example 1). Two plasmid constructs were transfected into EXPI293 cells by cationic lipofection at empirically determined ratios. The transfected cultures were incubated in cell culture medium for 6-8 days. After this, the cells were removed by centrifugation. This supernatant (medium, lane 1 of figure 13) contains construct 5 secreted into the medium by the transfected cells. Contaminating host cell proteins are also present in the culture medium. Construct 5 was purified from this medium by protein-a affinity chromatography. In this regard, the medium contains the desired construct 5 having three Fc domains (trimers) and a proportion of misassembled proteins containing two Fc domains (dimers, about 10% -15%) and one Fc domain (monomers, 5% -10%). There is still a small amount of contaminating host cell proteins. The protein a column eluate is buffer exchanged, concentrated and fractionated by strong cation exchanger (SCX) chromatography. Briefly, construct 5 was bound to an SCX column and then eluted with a salt and pH gradient. This step enables the isolation of the desired construct 5 with three Fc domains from most of the misfolded proteins with two or one Fc domain, from construct 5 with unwanted post-translational modifications, and from contaminating host cell proteins. After another round of concentration and buffer exchange, the final pure protein product of one construct 5 was obtained (pure, lane 2 of fig. 13).
FIG. 13 depicts SDS-PAGE of media obtained from cultured host cells engineered to express construct 5 (lane 1) and purified construct 5 (lane 2). A table showing the percentage of the major bands of SDS-PAGE for each sample is also shown. In this medium sample (lane 1), a major band of about 150kDa was observed, corresponding to the final protein product of construct 5 with three Fc domains. The medium sample also contained one minor band with weaker signal strength at 100kDa corresponding to a protein with two Fc domains, and one second minor band with the weakest signal strength at 50kDa corresponding to a protein with one Fc domain. After purification (lane 2), the final protein product of construct 5 with three Fc domains was enriched for one major band of approximately 150 kDa. Quantification of protein band signal intensity on SDS-PAGE of construct 5 showed that approximately 79% of the total protein was the desired protein product of construct 5 in the culture medium prior to protein purification. After protein purification, a substantially homologous set of constructs 5 with a purity of about 95% was obtained.
These findings confirm that, at CH3 a selective dimerization module in the antibody constant domain containing an engineered protrusion or an engineered cavity reduces self-association and prevents uncontrolled Fc-mediated aggregate or multimer formation, indicating that the dimerization selective module used in the constructs described herein can be used to generate a substantially homogeneous preparation of Fc constructs. This observation has a significant impact on the advantages in the manufacture, yield and purity of the constructs, for example, in order to control biological activity and potency.
Example 5 binding affinity and avidity
Cell-based FRET competition assays (Cisbio bioassays) were used to assess binding of constructs to multiple Fc γ receptors. Constructs 5 and 6 showed at least a tenfold reduction in IC50 (i.e., enhanced binding) for Fc γ RIIa, Fc γ RIIbn, and Fc γ RIIIa relative to the wild-type Fc domain (construct 1).
Example 6 monocyte activation and blockade assay
Three Fc constructs, construct 1, construct 5 and construct 6, containing one, three and two Fc domains respectively, were tested for their ability to activate THP-1 monocytes themselves. IL-8 release was used as an indicator of monocyte activation. Constructs 1, 5 and 6 were expressed and purified as described in examples 1 and 2. Each purified Fc construct was added to THP-1 monocytes. No significant IL-8 release was observed for any of the three constructs. This data is provided in fig. 14A.
The same three Fc constructs were then tested for their ability to inhibit Fc receptor-mediated monocyte activation. IgG1 (100. mu.g/mL) was immobilized on 96-well plates and used to induce IL-8 release by THP-1 monocytes. Serial dilutions of construct 1, construct 5 and construct 6 or control (intravenous immunoglobulin (IVIg), Human Serum Albumin (HSA), and glycine buffer) were then performed in tissue culture plates. Immediately add THP-1 monocytes (1.5X 10)5Individual cells) were mixed thoroughly at the same time. The culture was incubated for 18h and the supernatant was analyzed for IL-8. Construct 5 and construct 6 were found to inhibit IL-8 release more effectively than construct 1 at low doses. This data is provided in fig. 14B.
Example 7-K/BxN arthritis model
Fc construct 1, Fc construct 5 and Fc construct 6, as well as IVIg, were tested in the K/BxN serotransmission model using a method described in antoniy (Anthony), proces academy of sciences usa (proc.natl.acad.sci.u.s.a)105:19571-19578 (2008). K/BxN mice, 12 weeks old, were obtained/purchased from Jackson Laboratories. A total of 30C 57BL mice were divided into five groups of 6 mice each. One hour prior to injection of 200 μ l K/BxN serum, an arthritis inducing serum, each group (day 0) was injected intravenously (i.v.) with 200 μ l of 0.1g/ kg construct 6, 200 μ l of 0.1g/ kg construct 5, 200 μ l of 0.1g/kg IVIg, 230 μ l of 0.1g/kg IVIg, or 200 μ l of Phosphate Buffered Saline (PBS). Inflammation was scored by clinical examination of foot swelling and ankle thickness. For paw swelling, each paw was scored as 0-3 points (0, no swelling; 3, maximum swelling). Scores of four feet were added for the total clinical score of each individual mouse. For ankle thickness, caliper measurements were used. Each mouse was scored daily from day 0 to day 10. The daily mean clinical scores of six mice per group are plotted in figure 15. As shown in FIG. 15, 1g/kg of IVIg, 0.1g/kg of construct 5, and 0.1g/kg of construct 6 provided similar levels of inflammatory protection. Given that constructs 5 and 6 were administered at a dose ten times lower than the dose of IVIg, constructs 5 and 6 appeared to be more effective than IVIg.
Example 8 Chronic ITP model
Constructs 1 and 5 were tested for their ability to treat mice suffering from Immune Thrombocytopenia (ITP) as well as IVIg. ITP is induced by an antiplatelet Ab that causes platelet depletion. 45C 57BL/6 mice (18-22g, Charles river laboratories, MA, Mass.) were i.p. injected daily with 1.5. mu.g/mouse of rat anti-CD 41 antibody (Ab) (clone MWReg30 BioLegend Corp. catalog #133910) for 4 days (on days 1, 2, 3 and 4). Normal platelet levels were determined by injecting 5 mice with 1.5 μ g/mouse rat IgG1, a k isotype control Ab (BioLegend catalog # 400414). Ab was injected in 100. mu.l PBS. After a third injection of anti-CD 41 Ab on day 3, all mice were dosed intravenously once with 200 μ l saline control, 1g/kg IVIg, 0.02, 0.03, 0.1 and 0.3g/kg construct 1, and 0.004, 0.02 and 0.1g/kg construct 5. Mice were bled on day 5 (24 h after the fourth anti-CD 41 Ab injection) to quantify total platelet levels by the VetScan instrument. All procedures were performed following the Animal Welfare Act (Animal Welfare Act) and the laboratory guidelines for Animal Care and Use (Guide for the Care and Use of laboratory animals).
As shown in figure 16, platelet levels significantly increased after therapeutic treatment with 0.02 and 0.1g/kg of construct 5 when compared to saline (by one-way analysis of variance using multiple comparative tests p < 0.0001). Platelet levels in these groups were similar to those in the normal isotype-treated group. Therapeutic treatment with 1g/kg of IVIg and 0.1 and 0.3g/kg of construct 1 also significantly increased platelet levels when compared to saline (p < 0.05;. p <0.01, respectively, by one-way analysis of variance using multiple comparative tests), but platelet levels in these groups were lower than in the 0.02 and 0.1g/kg of construct 5 treated groups. In this model, construct 5 appears to be about 50-fold more potent than IVIg.
Example 9-construct 5 shows enhanced binding and affinity to Fc γ R compared to IVIg
According to the same protocol as described in example 8, the encoding overhang-20-charge Fc2*(construct F in example 1)*) And a cavity Fc*(construct C in example 1)*) Two plasmid constructs of (5) for expression and purification of construct*. The binding properties of this construct to different Fc receptors were compared to IVIG in a Fluorescence Resonance Energy Transfer (FRET) competitive binding assay.
Construct 5*Shows overall binding characteristics similar to IVIg to different Fc γ -receptors (with the lowest binding affinity observed for Fc γ RIIb), but with greatly enhanced binding to all low affinity Fc γ rs when compared to IVIg. Enhanced binding to Fc γ R corresponds to higher avidity, which refers to the cumulative effect of the cumulative affinity of each individual binding interaction. Construct 5*Consistently lower IC50 values than IVIg IC50 values, indicating significantly enhanced binding to low affinity Fc γ R compared to single IgG molecules. For example, construct 5 compared to IVIg*Shows about 170-fold enhanced affinity for Fc γ RIIa (H131 variant), 55-fold enhanced affinity for Fc γ RIIb.
EXAMPLE 10 inhibition of phagocytosis in THP-1 monocytes
Testing of construct 5 in a phagocytosis model*And IVIg.
Phagocytosis is the process by which cells (phagocytes) engulf solid particles, such as bacteria, to form an internal vesicle called a phagosome. In the immune system, phagocytosis is a major mechanism for elimination of pathogens and cellular debris. Monocytes and macrophages are included in cells that specifically clear opsonized (antibody-coated) particles from the immune system by phagocytosis, a mechanism that relies largely on Fc γ R-mediated conjugation. However, in autoimmune diseases, phagocytic cells can be activated, leading to the deleterious release of pro-inflammatory cytokines and phagocytosis of other critical cells in the body. IVIg containing pooled, multivalent IgG antibodies extracted from the plasma of over one thousand blood donors is used for the treatment of autoimmune diseases.
In this assay system, fluorescently labeled antibody-coated latex beads (a mimic of the opsonized bacteria or virus) are delivered to THP-1 cells and allowed to settle in construct 5*And phagocytosis in the presence and absence of IVIg. At the end of the incubation period, any external fluorescence was quenched with trypan blue, and the amount of intracellular fluorescence was quantified by flow cytometry. All groups were normalized to their untreated controls (THP-1 cells only and latex beads). Results are representative of two independent experiments.
As shown in FIG. 17, phagocytosis of opsonized beads by THP-1 monocytes Using IVIg and construct 5*Treatment of both inhibited, but construct 5*Has an IC50 value about 100-fold lower than the IC50 value of IVIg. This indicates that the Fc constructs of the invention (e.g., construct 5)*) Can be used for treating autoimmune indications as well as other indications treatable with IVIg.
Sequence listing
<110> momentum pharmaceuticals
<120> compositions and methods relating to engineered Fc constructs
<130> 50937-012WO3
<150> US 62/081,923
<151> 2014-11-19
<150> US 61/987,863
<151> 2014-05-04
<160> 46
<170> PatentIn version 3.5
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<213> Intelligent (Homo sapiens)
<400> 29
gacaagaccc acacctgtcc gccttgccct gcccctgagc tgctgggagg ccccagcgtg 60
ttcctgttcc ccccaaagcc caaggacacc ctgatgatca gccggacccc cgaagtgacc 120
tgcgtggtgg tggacgtgtc ccacgaggac cctgaagtga agttcaattg gtacgtggac 180
ggcgtggaag tgcacaacgc caagaccaag cccagagagg aacagtacaa cagcacctac 240
cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa agaatacaag 300
tgcaaagtct ccaacaaggc cctgcctgcc cccatcgaga aaaccatcag caaggccaag 360
ggccagcccc gcgagcccca ggtgtacaca ctgcccccca gccgggacga gctgaccaag 420
aaccaggtgt ccctgacctg cctggtgaaa ggcttctacc ccagcgatat cgccgtggaa 480
tgggagagca acggccagcc cgagaacaac tacaagacca ccccccctgt gctggacagc 540
gacggctcat tcttcctgta cagcaagctg accgtggaca agagccggtg gcagcagggc 600
aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc 660
ctgagcctga gccccggcaa g 681
<210> 30
<211> 227
<212> PRT
<213> Intelligent people
<400> 30
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys
225
<210> 31
<211> 681
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 31
gacaagaccc acacctgtcc gccttgccct gcccctgagc tgctgggagg ccccagcgtg 60
ttcctgttcc ccccaaagcc caaggacacc ctgatgatca gccggacccc cgaagtgacc 120
tgcgtggtgg tggacgtgtc ccacgaggac cctgaagtga agttcaattg gtacgtggac 180
ggcgtggaag tgcacaacgc caagaccaag cccagagagg aacagtacaa cagcacctac 240
cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa agaatacaag 300
tgcaaagtct ccaacaaggc cctgcctgcc cccatcgaga aaaccatcag caaggccaag 360
ggccagcccc gcgagcccca ggtgtacaca ctgcccccct gccgggacga gctgaccaag 420
aaccaggtgt ccctgtggtg cctggtgaaa ggcttctacc ccagcgatat cgccgtggaa 480
tgggagagca acggccagcc cgagaacaac tacaagacca ccccccctgt gctggacagc 540
gacggctcat tcttcctgta cagcaagctg accgtggaca agagccggtg gcagcagggc 600
aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc 660
ctgagcctga gccccggcaa g 681
<210> 32
<211> 227
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 32
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys
225
<210> 33
<211> 681
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 33
gacaagaccc acacctgtcc gccttgccct gcccctgagc tgctgggagg ccccagcgtg 60
ttcctgttcc ccccaaagcc caaggacacc ctgatgatca gccggacccc cgaagtgacc 120
tgcgtggtgg tggacgtgtc ccacgaggac cctgaagtga agttcaattg gtacgtggac 180
ggcgtggaag tgcacaacgc caagaccaag cccagagagg aacagtacaa cagcacctac 240
cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa agaatacaag 300
tgcaaagtct ccaacaaggc cctgcctgcc cccatcgaga aaaccatcag caaggccaag 360
ggccagcccc gcgagcccca agtgtgtaca ctgcccccca gccgggacga gctgaccaag 420
aaccaggtgt ccctgagctg cgccgtgaaa ggcttctacc ccagcgatat cgccgtggaa 480
tgggagagca acggccagcc cgagaacaac tacaagacca ccccccctgt gctggacagc 540
gacggctcat tcttcctggt tagcaagctg accgtggaca agagccggtg gcagcagggc 600
aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc 660
ctgagcctga gccccggcaa g 681
<210> 34
<211> 227
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 34
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys
225
<210> 35
<211> 681
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 35
gacaagaccc acacctgtcc gccttgccct gcccctgagc tgctgggagg ccccagcgtg 60
ttcctgttcc ccccaaagcc caaggacacc ctgatgatca gccggacccc cgaagtgacc 120
tgcgtggtgg tggacgtgtc ccacgaggac cctgaagtga agttcaattg gtacgtggac 180
ggcgtggaag tgcacaacgc caagaccaag cccagagagg aacagtacaa cagcacctac 240
cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa agaatacaag 300
tgcaaagtct ccaacaaggc cctgcctgcc cccatcgaga aaaccatcag caaggccaag 360
ggccagcccc gcgagcccca ggtgtacaca ctgcccccca gccgggacga gctgaccaag 420
aaccaggtgt ccctgacctg cctggtgaaa ggcttctacc ccagcgatat cgccgtggaa 480
tgggagagca acggccagcc cgagaacaac tacaagacca ccccccctgt gctgaaaagc 540
gacggctcat tcttcctgta cagcgacctg accgtggaca agagccggtg gcagcagggc 600
aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc 660
ctgagcctga gccccggcaa g 681
<210> 36
<211> 227
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 36
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys
225
<210> 37
<211> 1398
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 37
gacaagaccc acacctgtcc cccttgccct gcccctgagc tgctgggagg ccccagcgtg 60
ttcctgttcc ccccaaagcc caaggacacc ctgatgatca gccggacccc cgaagtgacc 120
tgcgtggtgg tggacgtgtc ccacgaggac cctgaagtga agttcaattg gtacgtggac 180
ggcgtggaag tgcacaacgc caagaccaag cccagagagg aacagtacaa cagcacctac 240
cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tcaacggcaa agagtacaag 300
tgcaaggtgt ccaacaaggc cctgcctgcc cccatcgaga aaaccatcag caaggccaag 360
ggccagcccc gcgagcccca ggtctacaca ctgcccccca gccgggacga gctgaccaag 420
aaccaggtct ccctgacctg cctggtgaaa ggcttctacc ccagcgatat cgccgtggaa 480
tgggagagca acggccagcc cgagaacaac tacaagacca ccccccctgt gctggacagc 540
gacggctcat tcttcctgta cagcaagctg accgtggaca agagccggtg gcagcagggc 600
aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc 660
ctgagcctga gccccggcaa aggcggggga tctgggggag gaagcggagg cggcagcgat 720
aagacccata cctgccctcc ctgtcccgct cccgaactgc tggggggacc ctccgtgttt 780
ctgtttccac ctaagcctaa ggatacgctc atgatctcca gaacccctga agtcacatgt 840
gtggtggtcg atgtgtctca tgaagatccc gaagtcaagt ttaactggta tgtggatggg 900
gtcgaggtcc acaatgccaa aacaaagcct cgggaagaac agtataactc cacctacaga 960
gtcgtcagcg tgctgacagt ccttcatcag gattggctga atgggaaaga gtacaaatgt 1020
aaagtgtcta acaaagctct gcccgctcct atcgaaaaga ccatctccaa agccaaaggg 1080
cagcccagag aacctcaggt gtacaccctg ccaccctcca gagatgagct gacaaaaaat 1140
caggtgtcac tgacatgtct ggtgaaaggg ttttatccct ccgacattgc tgtggaatgg 1200
gaatccaatg ggcagcctga aaacaattat aagacaacac ctcccgtgct ggactccgat 1260
ggctcatttt ttctgtactc taaactgaca gtggataagt ccagatggca gcagggaaat 1320
gtgttttcct gctctgtgat gcatgaagct ctgcataatc actatacaca gaaaagcctg 1380
tccctgtccc ccggcaag 1398
<210> 38
<211> 466
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 38
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Asp
225 230 235 240
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
245 250 255
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
260 265 270
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
275 280 285
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
290 295 300
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
305 310 315 320
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
325 330 335
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
340 345 350
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
355 360 365
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
370 375 380
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
385 390 395 400
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
405 410 415
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
420 425 430
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
435 440 445
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
450 455 460
Gly Lys
465
<210> 39
<211> 1422
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 39
gacaagaccc acacctgtcc cccttgccca gcccctgagc tgctgggagg ccccagcgtg 60
ttcctgttcc ccccaaagcc caaggacacc ctgatgatca gccggacccc cgaagtgacc 120
tgcgtggtgg tggacgtgtc ccacgaggac cctgaagtga agttcaattg gtacgtggac 180
ggcgtggaag tgcacaacgc caagaccaag cccagagagg aacagtacaa cagcacctac 240
cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa agagtacaag 300
tgcaaggtgt ccaacaaggc cctgcctgcc cccatcgaga aaaccatcag caaggccaag 360
ggccagcccc gcgagcccca ggtgtacacc ctgccccctt gcagagatga gctgaccaag 420
aaccaggtgt ccctgtggtg cctggtcaag ggcttctacc ccagcgatat cgccgtggaa 480
tgggagagca acggccagcc cgagaacaac tacaagacca ccccccctgt gctggacagc 540
gacggctcat tcttcctgta cagcaagctg accgtggaca agagccggtg gcagcagggc 600
aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc 660
ctgagcctga gccccggcaa gtctggggga ggatcagggg gtggaagtgg cggtggatct 720
ggtggtggaa gcggaggcgg cgataagaca cacacatgcc ccccctgtcc agctcccgaa 780
ctgctggggg gaccctccgt gtttctgttt ccacctaagc ctaaggatac gctcatgatc 840
tccagaaccc ctgaagtcac atgtgtggtg gtcgatgtgt ctcatgaaga tcccgaagtc 900
aagtttaatt ggtatgtcga tggggtcgag gtgcacaatg ccaaaacaaa acctcgggaa 960
gaacagtata actccacata cagagtggtg tctgtcctca cagtcctgca tcaggattgg 1020
ctcaatggga aagagtacaa atgtaaagtc tctaacaagg ctctccccgc tccgatcgaa 1080
aagaccatct ccaaagccaa agggcagccc agagaacctc aggtctacac actgcctccc 1140
agccgggacg agctgacaaa aaatcaagtg tctctgacct gcctcgtgaa gggcttttat 1200
ccctccgaca ttgccgtcga gtgggagtcc aatggacagc cggaaaacaa ttataagacc 1260
acgcctccag tgctgaagtc cgacggcagc ttctttctgt actccgacct gacagtggat 1320
aagtccagat ggcagcaagg gaatgtgttc tcctgttccg tgatgcatga agccctccat 1380
aatcactata cccagaaaag cctgtccctg tcccctggca ag 1422
<210> 40
<211> 474
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 40
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
225 230 235 240
Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys
245 250 255
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
260 265 270
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
275 280 285
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
290 295 300
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
305 310 315 320
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
325 330 335
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
340 345 350
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
355 360 365
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
370 375 380
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
385 390 395 400
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
405 410 415
Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe
420 425 430
Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
435 440 445
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
450 455 460
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
465 470
<210> 41
<211> 1422
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 41
gacaagaccc acacctgtcc cccttgccct gcccctgagc tgctgggagg ccccagcgtg 60
ttcctgttcc ccccaaagcc caaggacacc ctgatgatca gccggacccc cgaagtgacc 120
tgcgtggtgg tggacgtgtc ccacgaggac cctgaagtga agttcaattg gtacgtggac 180
ggcgtggaag tgcacaacgc caagaccaag cccagagagg aacagtacaa cagcacctac 240
cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa agagtacaag 300
tgcaaggtgt ccaacaaggc cctgcctgcc cccatcgaga aaaccatcag caaggccaag 360
ggccagcccc gcgagcccca ggtgtacacc ctgccccctt gcagagatga actgaccaag 420
aaccaggtgt ccctgtggtg cctggtcaag ggcttctacc ccagcgatat cgccgtggaa 480
tgggagagca acggccagcc cgagaacaac tacaagacca ccccccctgt gctggacagc 540
gacggctcat tcttcctgta cagcaagctg accgtggaca agagccggtg gcagcagggc 600
aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc 660
ctgagcctga gccccggcaa gtctggggga ggatcagggg gtggaagtgg cggtggatct 720
ggtggtggaa gcggaggcgg cgataagaca cacacatgcc ccccctgtcc agctcccgaa 780
ctgctggggg gaccctccgt gtttctgttt ccacctaagc ctaaggatac gctcatgatc 840
tccagaaccc ctgaagtcac atgtgtggtg gtcgatgtgt ctcatgaaga tcccgaagtc 900
aagtttaact ggtatgtgga tggggtcgag gtccacaatg ccaaaacaaa gcctcgggaa 960
gaacagtata actccaccta cagagtcgtc agcgtgctga cagtcctgca tcaagattgg 1020
ctcaatggga aagagtataa gtgtaaagtc tcgaacaaag ccctccccgc tcctatcgaa 1080
aagaccatct ccaaagccaa agggcagccc agagaacctc aggtctacac actgcctcca 1140
tgtcgggacg agctgacaaa aaatcaggtg tcactgtggt gtctggtgaa ggggttttac 1200
ccttccgaca ttgctgtgga atgggaatcc aatgggcagc ctgaaaacaa ttataagaca 1260
acacctcccg tgctggactc cgatggctca ttttttctgt actctaaact gacagtggat 1320
aagtccagat ggcagcaggg aaatgtgttt tcctgctctg tgatgcatga agctctgcat 1380
aatcactata cacagaaaag cctgtccctg tcccctggca ag 1422
<210> 42
<211> 474
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 42
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
225 230 235 240
Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys
245 250 255
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
260 265 270
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
275 280 285
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
290 295 300
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
305 310 315 320
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
325 330 335
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
340 345 350
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
355 360 365
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu
370 375 380
Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr
385 390 395 400
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
405 410 415
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
420 425 430
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
435 440 445
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
450 455 460
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
465 470
<210> 43
<211> 1365
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 43
aggacagtgg ccgctcccag cgtgttcatc ttcccaccca gcgacgagca gctgaagtcc 60
ggcacagcca gcgtggtctg cctgctgaac aacttctacc cccgcgaggc caaggtgcag 120
tggaaggtgg acaacgccct gcagagcggc aacagccagg aaagcgtcac cgagcaggac 180
agcaaggact ccacctacag cctgtctagc accctgaccc tgagcaaggc cgactacgag 240
aagcacaagg tgtacgcctg cgaagtgacc caccagggcc tgtccagccc cgtgaccaag 300
agcttcaaca gaggcgagtg cggcggctct ggcggaggat ccgggggagg atcaggcggc 360
ggaagcggag gcagcgctag cacaaagggc ccctccgtgt tccccctggc ccccagcagc 420
aagagcacat ctggcggaac agccgccctg ggctgcctgg tgaaagacta cttccccgag 480
cccgtgaccg tgtcctggaa ctctggcgcc ctgaccagcg gcgtgcacac ctttccagcc 540
gtgctgcaga gcagcggcct gtactccctg agcagcgtgg tgacagtgcc tagcagcagc 600
ctgggcaccc agacctacat ctgcaacgtg aaccacaagc ccagcaacac caaagtggac 660
aagcgggtgg aacccaagag ctgcgacaag acccacacgt gtcccccctg cccagcccct 720
gaactgctgg gcggacctag cgtgttcctg ttccccccaa agcccaagga caccctgatg 780
atcagccgga cccccgaagt gacctgcgtg gtggtggacg tgtcccacga ggaccctgaa 840
gtgaagttca attggtacgt ggacggcgtg gaagtgcaca atgccaagac caagcccaga 900
gaggaacagt acaacagcac ctaccgggtg gtgtccgtgc tgaccgtgct gcaccaggac 960
tggctgaacg gcaaagagta caagtgcaag gtctccaaca aggccctgcc tgcccccatc 1020
gagaaaacca tcagcaaggc caagggccag ccccgcgagc cccaggtgta cacactgccc 1080
cccagccggg acgagctgac caagaaccag gtgtccctga cctgtctggt gaaaggcttc 1140
tacccctccg atatcgccgt ggaatgggag agcaacggcc agcccgagaa caactacaag 1200
accacccccc ctgtgctgga ctccgacggc tcattcttcc tgtacagcaa gctgaccgtg 1260
gacaagagcc ggtggcagca gggcaacgtg ttcagctgct ccgtgatgca cgaggccctg 1320
cacaaccact acacccagaa gtccctgagc ctgagccccg gcaaa 1365
<210> 44
<211> 455
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 44
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Ser Gly Gly
100 105 110
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Ser Ala Ser Thr
115 120 125
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
130 135 140
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
145 150 155 160
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
165 170 175
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
195 200 205
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu
210 215 220
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
225 230 235 240
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
355 360 365
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
385 390 395 400
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
435 440 445
Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 45
<211> 227
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 45
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Ser Cys Ala Val Glu Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys
225
<210> 46
<211> 474
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic construct
<400> 46
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
225 230 235 240
Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys
245 250 255
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
260 265 270
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
275 280 285
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
290 295 300
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
305 310 315 320
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
325 330 335
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
340 345 350
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
355 360 365
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
370 375 380
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
385 390 395 400
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
405 410 415
Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe
420 425 430
Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
435 440 445
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
450 455 460
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
465 470

Claims (17)

1. An Fc construct comprising:
a) a first polypeptide having the formula a-L-B; wherein
i) A comprises a first Fc domain monomer;
ii) L is a linker; and is
B comprises a second Fc domain monomer;
b) a second polypeptide having the formula a ' -L ' -B '; wherein
i) A' comprises a third Fc domain monomer;
l' is a linker; and is
B' comprises a fourth Fc domain monomer;
c) a third polypeptide comprising a fifth Fc domain monomer; and
d) a fourth polypeptide comprising a sixth Fc domain monomer;
wherein the Fc construct contains no more than three Fc domains;
wherein the Fc domain monomers are all IgG1 Fc domain monomers;
wherein the first polypeptide and the second polypeptide have the same amino acid sequence and wherein the third polypeptide and the fourth polypeptide have the same amino acid sequence; and
wherein A and A 'combine to form a first Fc domain, B and the fifth Fc domain monomer combine to form a second Fc domain, and B' and the sixth Fc domain monomer combine to form a third Fc domain,
wherein:
the first and third Fc domain monomers each comprise complementary dimerization selectivity modules that promote dimerization between the first and third Fc domain monomers, wherein the complementary dimerization selectivity modules of the first and third Fc domain monomers each comprise opposing charge mutations at least two positions,
the second and fifth Fc domain monomers each comprise complementary dimerization selectivity modules that promote dimerization between the second and fifth Fc domain monomers, wherein one of the complementary dimerization selectivity modules of the second and fifth Fc domain monomers comprises an engineered protrusion and the other of the complementary dimerization selectivity modules of the second and fifth Fc domain monomers comprises an engineered cavity, an
The fourth Fc domain monomer and the sixth Fc domain monomer each comprise complementary dimerization selectivity modules that promote dimerization between the fourth Fc domain monomer and the sixth Fc domain monomer, wherein one of the complementary dimerization selectivity modules of the fourth Fc domain monomer and the sixth Fc domain monomer comprises an engineered protrusion and the other of the complementary dimerization selectivity modules of the fourth Fc domain monomer and the sixth Fc domain monomer comprises an engineered cavity.
2. The Fc construct of claim 1, wherein the opposite charge mutations at said at least two positions are K409D/D399K, K392D/D399K, E357K/K370E, D356K/K439D, K409E/D399K, K392E/D399K, E357K/K370D, or D356K/K439E.
3. The Fc construct of claim 1, wherein complementary dimerization selectivity modules of said first Fc domain monomer and said third Fc domain monomer are comprised at CH3 domain at the interface between four positions in four opposite charge mutation.
4. The Fc construct of claim 3, wherein said four oppositely charged mutations combine double mutations of any pair selected from the group consisting of K409D/D399K, K392D/D399K, E357K/K370E, D356K/K439D, K409E/D399K, K392E/D399K, E357K/K370D, and D356K/K439E.
5. The Fc construct of claim 1, wherein said complementary dimerization selectivity modules of the second and fourth Fc domain monomers each comprise an engineered protrusion and said complementary dimerization selectivity modules of the fifth and sixth Fc domain monomers each comprise an engineered cavity.
6. The Fc construct of claim 5, wherein said engineered protrusion comprises at least one mutation selected from S354C, T366W, T366Y, T394W, T394F, and F405W, and said engineered cavity comprises at least one mutation selected from Y349C, T366S, L368A, Y407V, Y407T, Y407A, F405A, and T394S.
7. The Fc construct of claim 1, wherein said complementary dimerization selectivity modules of the second and fourth Fc domain monomers each comprise an engineered cavity, and said complementary dimerization selectivity modules of the fifth and sixth Fc domain monomers each comprise an engineered protrusion.
8. The Fc construct of claim 7, wherein said engineered cavity comprises at least one mutation selected from Y349C, T366S, L368A, Y407V, Y407T, Y407A, F405A, and T394S, and said engineered protuberance comprises at least one mutation selected from S354C, T366W, T366Y, T394W, T394F, and F405W.
9. The Fc construct of claim 1, wherein the complementary dimerization selectivity module of said first Fc domain monomer is comprised at CH3 domain, the complementary dimerization selectivity module of the third Fc domain monomer comprises mutations of opposite charge at least two positions within the loop of charged residues at the interface between the domainsH3 at least two positions within the loop of charged residues at the interface between the domains, and a second FcThe complementary dimerization selectivity module of the domain monomer comprises an engineered protrusion, the complementary dimerization selectivity module of the fifth Fc domain monomer comprises an engineered cavity, the complementary dimerization selectivity module of the fourth Fc domain monomer comprises an engineered protrusion, and the complementary dimerization selectivity module of the sixth Fc domain monomer comprises an engineered cavity.
10. The Fc construct of claim 1, wherein said complementary dimerization selectivity module of the first Fc domain monomer comprises an engineered protrusion, said complementary dimerization selectivity module of the third Fc domain monomer comprises an engineered cavity, and said complementary dimerization selectivity module of the second Fc domain monomer comprises a moiety at CH3 domain, the complementary dimerization selectivity module of the fifth Fc domain monomer comprises mutations of opposite charge at least two positions within the loop of charged residues at the interface between the domainsH3 domain, and complementary dimerization selectivity modules of the fourth Fc domain monomer comprise opposite charge mutations at least two positions within the loop of charged residues at the interface between the domainsH3 at least two positions within the loop of charged residues at the interface between the domains, and the complementary dimerization selectivity module of the sixth Fc domain monomer is comprised at CH3 domain at the interface between the charged residues in the ring at least two positions of opposite charge mutation.
11. The Fc construct of claim 1, wherein said complementary dimerization selectivity module of the first Fc domain monomer comprises an engineered cavity, said complementary dimerization selectivity module of the third Fc domain monomer comprises an engineered protrusion, and said complementary dimerization selectivity module of the second Fc domain monomer comprises a functional group at CH3 domain, the complementary dimerization selectivity module of the fifth Fc domain monomer comprises mutations of opposite charge at least two positions within the loop of charged residues at the interface between the domainsH3 at the interface between the domains, at least two positions within the loop of charged residues, andthe complementary dimerization selectivity module of the fourth Fc domain monomer is contained in CH3 at least two positions within the loop of charged residues at the interface between the domains, and the complementary dimerization selectivity module of the sixth Fc domain monomer is comprised at CH3 domain at the interface between the charged residues in the ring at least two positions of opposite charge mutation.
12. The Fc construct of claim 1, wherein one or more linkers in said Fc construct is a spacer.
13. A pharmaceutical composition comprising a substantially homogeneous collection of Fc constructs of claim 1 and one or more pharmaceutically acceptable carriers or excipients.
14. Use of the Fc construct of claim 1 in the manufacture of a medicament for reducing immune cell activation of an immune response in a subject, wherein the Fc construct is formulated for administration to a subject.
15. The use of claim 14, wherein the subject has an autoimmune disease.
16. Use of an Fc construct of claim 1 in the manufacture of a medicament for treating inflammation in a subject, wherein the Fc construct is formulated for administration to a subject.
17. Use of an Fc construct of claim 1 in the manufacture of a medicament for promoting clearance of autoantibodies and/or inhibiting antigen presentation in a subject, wherein the Fc construct is formulated for administration to a subject.
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